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.

IMAGING OF VITELLIFORM MACULAR LESIONS USING POLARIZATION-SENSITIVE OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To examine the involvement of the retinal pigment epithelium (RPE) in the presence of vitelliform macular lesions (VML) in Best vitelliform macular dystrophy (BVMD), autosomal recessive bestrophinopathy, and adult-onset vitelliform macular degeneration using polarization-sensitive optical coherence tomography (PS-OCT). METHODS: A total of 35 eyes of 18 patients were imaged using a PS-OCT system and blue light fundus autofluorescence imaging. Pathogenic mutations in the BEST1 gene, 3 of which were new, were detected in all patients with BVMD and autosomal recessive bestrophinopathy. RESULTS: Polarization-sensitive optical coherence tomography showed a characteristic pattern in all three diseases with nondepolarizing material in the subretinal space consistent with the yellowish VML seen on funduscopy with a visible RPE line below it. A focal RPE thickening was seen in 26 eyes under or at the edge of the VML. Retinal pigment epithelium thickness outside the VML was normal or mildly thinned in patients with BVMD and adult-onset vitelliform macular degeneration but was diffusely thinned or atrophic in patients with autosomal recessive bestrophinopathy. Patients with autosomal recessive bestrophinopathy showed sub-RPE fibrosis alongside the subretinal VML. Polarization-sensitive optical coherence tomography was more reliable in assessing the localization and the integrity of the RPE than spectral domain OCT alone. On spectral domain OCT, identification of the RPE was not possible in 19.4% of eyes. Polarization-sensitive optical coherence tomography allowed for definite identification of the location of VML in respect to the RPE in all eyes, since it provides a tissue-specific contrast. CONCLUSION: Polarization-sensitive optical coherence tomography confirms in vivo the subretinal location of VML and is useful in the assessment of RPE integrity.

NOVEL BEST1 MUTATIONS DETECTED BY NEXT-GENERATION SEQUENCING IN A CHINESE POPULATION WITH VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To characterize novel BEST1 mutations and the phenotype-genotype correlations in vitelliform macular dystrophy in a Chinese population. METHODS: Seventeen individuals affected by vitelliform macular dystrophy underwent detailed ophthalmic examinations, including a best-corrected visual acuity test, slit-lamp biomicroscopy, fundus photography, spectral-domain optical coherence tomography, fundus autofluorescence, fluorescein angiography, and optical coherence tomography angiography. Next-generation sequencing was used to screen 790 genes frequently involved in common inherited nonsyndromic eye diseases in all participants. Sanger sequencing was used to identify possible disease-causing variants. RESULTS: The mean ± SD age of the patients was 44.20 ± 15.09 (range: 16-69) years. Seven novel BEST1 mutations were identified: six heterozygous missense (p.Thr307Asn, p.Ile295The, p.Leu75Pro, p.Thr2Ser, p.Ser79Tyr, and p.Val81Leu) and one frameshift (p.Glu115GlufsX120) mutation. Choroidal neovascularization was detected in two probands. One individual presented with subfoveal focal choroidal excavation. Arden ratios obtained by electrooculography were less than the 1.5 cutoff value in 7 patients. No mutations were identified in 2 patients, one of whom had a fundus appearance typical of vitelliform macular dystrophy and a decreased Arden ratio (1.2/1.2). CONCLUSION: Patients with the same heterozygous BEST1 mutations exhibited varying phenotypes. Our results have expanded the BEST1 mutation spectrum in a Chinese population with vitelliform macular dystrophy.

THE FUNDUS PHENOTYPE ASSOCIATED WITH THE p.Ala243Val BEST1 MUTATION.

PURPOSE: To describe a highly recognizable and reproducible retinal phenotype associated with a specific BEST1 mutation-p.Ala243Val. METHODS: Retrospective review of consecutive cases where genetic testing has identified p.Ala243Val BEST1 as the cause of disease. Electronic patient records were used to extract demographic, as well as functional and anatomical data. These data were compared with those observed with the most common BEST1 genotype, p.Arg218Cys. RESULTS: Eight individuals (six families) were identified with the p.Ala243Val BEST1 mutation and seven patients with the pathologic variant p.Arg218Cys. No patients with mutation of codon 243 knowingly had a family history of retinal disease, whereas all patients with the p.Arg218Cys variant did. The maculopathy was bilateral in all cases. The p.Ala243Val mutation was associated with a pattern dystrophy-type appearance, most visible with near-infrared reflectance and fundus autofluorescence imaging. This phenotype was never observed with any other genotype. This mutation was associated with an older median age of symptom onset (median = 42, interquartile range = 22) compared with those harboring the p.Arg218Cys mutation (median = 18, interquartile range = 12; Mann-Whitney U test; P < 0.05). Despite their older age, the final recorded acuity seemed to be better in the p.Ala243Val group (median = 0.55, interquartile range = 0.6475; median = 0.33, interquartile range = 0.358), although this did not reach statistical significance (Mann-Whitney U test; P > 0.05). CONCLUSION: The mutation p.Ala243Val is associated with highly recognizable and reproducible pattern dystrophy-like phenotype. Patients develop symptoms at a later age and tend to have better preservation of electrooculogram amplitudes.

SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY FEATURES IN DIFFERENT STAGES OF BEST VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To provide a systematic classification of findings regarding the different stages of vitelliform macular dystrophy on spectral domain optical coherence tomography (SD-OCT). METHODS: Ninety-four eyes of 47 patients were recruited in a prospective cross-sectional study. All patients underwent a complete ophthalmologic examination, including best-corrected visual acuity using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, biomicroscopy, and SD-OCT. The findings assessed included vitelliform material, neurosensory detachment, status of external limiting membrane, ellipsoid zone and retinal pigment epithelium, choroidal excavation, foveal cavitation, choroidal neovascularization, vitreomacular traction, and macular hole. The primary outcome measure was the identification of SD-OCT findings in each vitelliform macular dystrophy stage. Secondary outcomes included the correlations between SD-OCT features and visual acuity changes. RESULTS: The outer retinal layers (external limiting membrane, ellipsoid zone, and retinal pigment epithelium) were found to be more commonly disrupted in Stages 2 to 4 (range: 86%-100%), whereas their absence was more typical of Stage 5 (71%-86%). Vitelliform material was found in 100% of Stages 2 and 3, 93% of Stage 4, and interestingly in 43% of Stage 5. Eyes characterized by vitelliform material showed a greater correlation with higher best-corrected visual acuity than eyes without it (0.35 logarithm of the minimum angle of resolution vs. 0.80 ± 0.36 logarithm of the minimum angle of resolution, approximately 20/45 and 20/125 Snellen equivalent, respectively) (t = 3.726, P < 0.05). Moreover, its absence was associated with a best-corrected visual acuity of 0.5 logarithm of the minimum angle of resolution or worse (approximately 20/63 Snellen equivalent; P < 0.05). Subretinal fluid was more common in Stages 3 and 4 (72.7% and 75%, respectively) than Stages 2 and 5 (P = 0.004). Eyes with subretinal fluid were significantly associated with a visual acuity of 0.2 logarithm of the minimum angle of resolution or worse (approximately 20/32 Snellen equivalent; P = 0.04). CONCLUSION: Spectral domain optical coherence tomography assessment primarily indicates an outer retinal layer disruption in Stages 2 to 4, along with the presence of vitelliform material extending into the more advanced clinical stages too. Eyes characterized by the persistence of vitelliform material show better best-corrected visual acuity. Future investigations based on a longitudinal follow-up are warranted to correlate SD-OCT modifications with functional responses to identify SD-OCT indicators for prognostic and therapeutic purposes.

MICROPERIMETRY IN BEST VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To investigate retinal sensitivity in eyes with all the clinical stages of Best vitelliform macular dystrophy (VMD). METHODS: Thirty-two patients affected by VMD in subclinical, vitelliform, pseudohypopyon, vitelliruptive, and atrophic stages were enrolled in this prospective cross-sectional study. Patients underwent a complete ophthalmologic examination, including determination of best-corrected visual acuity (BCVA), staging of the disease (Gass's classification), and microperimetry by means of the macular integrity assessment microperimeter. The primary outcome measure was to describe the alterations in the retinal sensitivity of eyes affected by VMD in different stages. Secondary outcome measures included correlations between retinal sensitivity and best-corrected visual acuity and the correlation between the VMD stage and the specific microperimetry pattern. RESULTS: Mean retinal sensitivity was reduced in all the VMD stages. Nevertheless, vitelliform, pseudohypopyon, and vitelliruptive stages turned out to be very similar, especially within 10°. Fixation was classified as stable in 27 eyes (44.2%), relatively unstable in 16 eyes (26.2%), and unstable in 18 eyes (29.5%). Fixation stability correlated both with the disease stage and best-corrected visual acuity. CONCLUSION: VMD is characterized by complex microperimetric abnormalities, involving the whole macular area. Microperimetry may contribute to the global clinical assessment of patients affected by VMD and could be used in future therapeutic approaches.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN ADULT-ONSET FOVEOMACULAR VITELLIFORM DYSTROPHY.

PURPOSE: To determine the ability of optical coherence tomography angiography (OCTA) to detect choroidal neovascularization (CNV) in the pseudohypopyon stage of adult-onset foveomacular vitelliform dystrophy. METHODS: Prospective case series of eight consecutive patients with adult-onset foveomacular vitelliform dystrophy with at least one eye in the pseudohypopyon stage (a total of 14 eyes). Patients were assessed with spectral domain OCT, flourescein angiography, and OCTA. Main outcome measures were the presence or absence of CNV and any unifying patterns that could be identified on OCTA for adult-onset foveomacular vitelliform dystrophy. RESULTS: One (12.5%) of eight eyes in the pseudohypopyon stage had CNV on OCTA, without definitive evidence of CNV on flourescein angiography. Twelve of 14 eyes (86%) had OCTA segmentation errors, giving the false appearance of deep capillary plexus drop out. All 14 eyes (100%) had blockage of flow signal under the vitelliform lesion on OCTA that presented as artifactual loss of flow in the choriocapillaris. CONCLUSION: Optical coherence tomography angiography may be superior to flourescein angiography in detecting CNV in adult-onset foveomacular vitelliform dystrophy, especially in the pseudohypopyon stage. There are common artifacts that must be considered when analyzing vitelliform lesions with OCTA, including segmentation errors and inability to visualize flow under the vitelliform lesion in the choriocapillaris.

ADULT-ONSET FOVEOMACULAR VITELLIFORM DYSTROPHY EVALUATED BY MEANS OF OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY: A Comparison With Dry Age-Related Macular Degeneration and Healthy Eyes.

PURPOSE: To investigate alterations of superficial and deep retinal vascular densities, as well as of choroidal thickness, in patients affected by adult-onset foveomacular vitelliform dystrophy (AOFVD). METHODS: A total of 22 eyes (15 patients) affected by AOFVD were recruited in the study. Furthermore, 20 eyes of 20 healthy subjects and 20 eyes of 18 patients affected by intermediate dry age-related macular degeneration (AMD) were enrolled. All patients underwent a complete ophthalmologic examination, including optical coherence tomography angiography. Outcome measures were superficial vessel density, deep vessel density, and choroidal thickness. RESULTS: Parafoveal superficial vessel density was increased in patients with AOFVD compared with the AMD group (50.6 ± 4.3% and 46.3 ± 4.3%, respectively, P = 0.016). Parafoveal deep vessel density was 57.9 ± 6.4% in patients with AOFVD, 52.2 ± 3.8% in patients with AMD, and 52.7 ± 6.0% in healthy controls (P = 0.006 and P = 0.035, respectively, after comparison with the AOFVD group). CONCLUSION: We demonstrated that both superficial and deep vessel densities were significantly increased in patients with AOFVD, after the comparison with intermediate patients with AMD. These findings suggest that the pathogenic mechanisms in AOFVD are different from those in AMD and that optical coherence tomography angiography could be useful in differentiate early stages of these two diseases.

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.

Optical Coherence Tomography Examination of the Retinal Pigment Epithelium in Best Vitelliform Macular Dystrophy.

PURPOSE: To describe the anatomic changes and natural history of vitelliform lesions in Best vitelliform macular dystrophy (BVMD) using spectral-domain optical coherence tomography (OCT). DESIGN: Prospective comparative case series. PARTICIPANTS: Twenty patients (40 eyes) with molecular confirmation of mutation in the BEST1 gene and 20 age-matched controls were included. METHODS: Color fundus photographs, fundus autofluorescence, and spectral-domain OCT were obtained, and these findings were compared between the 2 groups. Fifteen of the 20 patients with Best disease had more than 1 visit, and the imaging studies from each visit were compared with each other over time. MAIN OUTCOME MEASURES: Evolution of visual acuity and clinical stage of BVMD correlated to OCT measurement parameters, including retinal pigment epithelium (RPE) thickness, central macular thickness, and integrity of the ellipsoid zone. RESULTS: Patients with BVMD demonstrated progressive disorganization and thinning of the submacular RPE on OCT when compared with normal controls. Concurrent with the appearance of "egg-yolk lesions," the OCT showed a cleft in the outer retina, creating an apical and basal separation of retinal layers. The apical complex of the vitelliform lesion eventually degenerated and flattened. Patients with such lesions nevertheless maintained reasonable visual acuity into the advanced vitelleruptive stages despite the disruption of normal anatomic changes. CONCLUSIONS: Our study suggests that in BVMD, subretinal vitelliform material accumulation leads to a clear separation of the outer retinal layers. The level at which this cleft forms is a topic of discussion and interest, with the most likely levels of least resistance being the interdigitation zone or between the RPE and the Bruch's membrane. It is possible that RPE may continue to form a preserved photoreceptor-RPE complex that provides essential nutrients to the photoreceptors and in turn helps patients maintain better than expected visual acuity for years.

PHOTORECEPTOR INNER SEGMENT MORPHOLOGY IN BEST VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To characterize outer retina structure in best vitelliform macular dystrophy (BVMD) and to determine the effect of macular lesions on overlying and adjacent photoreceptors. METHODS: Five individuals with BVMD were followed prospectively with spectral domain optical coherence tomography and confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). The AOSLO cone photoreceptor mosaic images were obtained within and around retinal lesions. Cone density was measured inside and outside lesions. In 2 subjects, densities were compared with published measurements acquired ∼2.5 years before. One subject was imaged 3 times over a 5-month period. RESULTS: The AOSLO imaging demonstrated that photoreceptor morphology within BVMD retinal lesions was highly variable depending on the disease stage, with photoreceptor structure present even in advanced disease. The AOSLO imaging was repeatable even in severe disease over short-time and long-time intervals. Photoreceptor density was normal in retinal areas immediately adjacent to lesions and stable over ∼2.5 years. Mobile disk-like structures possibly representing subretinal macrophages were also observed. CONCLUSION: Combined confocal and nonconfocal split-detector AOSLO imaging reveals substantial variability within clinical lesions in all stages of BVMD. Longitudinal cellular photoreceptor imaging could prove a powerful tool for understanding disease progression and monitoring emerging therapeutic treatment response in inherited degenerations such as BVMD.

Detailed analysis of family with autosomal recessive bestrophinopathy associated with new BEST1 mutation.

PURPOSE: To describe the clinical and genetic findings in a patient with autosomal recessive bestrophinopathy (ARB) and his healthy parents. METHODS: The patient and his healthy non-consanguineous parents underwent detailed ophthalmic evaluations including electro-oculography (EOG), spectral-domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF) imaging. Mutation analysis of the BEST1 gene was performed by Sanger sequencing. RESULTS: The FAF images showed multiple spots of increased autofluorescence, and the sites of these spots corresponded to the yellowish deposits detected by ophthalmoscopy. SD-OCT showed cystoid macular changes and a shallow serous macular detachment. The Arden ratio of the EOG was markedly reduced to 1.1 in both eyes. Genetic analysis of the proband detected two sequence variants of the BEST1 gene in the heterozygous state: a novel variant c.717delG, p.V239VfsX2 and an already described c.763C>T, p.R255W variant associated with Best vitelliform macular dystrophy and ARB. The proband's father carried the c.717delG, p.V239VfsX2 variant in the heterozygous state, and the mother carried the c.763C>T, p.R255W variant in the heterozygous state. The parents who were heterozygous for the BEST1 variants had normal visual acuity, EOG, SD-OCT, and FAF images. CONCLUSIONS: In a truncating BEST1 mutation, the phenotype associated with ARB is most likely due to a marked decrease in the expression of BEST1 promoted by the nonsense-mediated decay surveillance mechanism, and it may depend on the position of the premature termination of the codon created.

Retinal structure in young patients aged 10 years or less with Best vitelliform macular dystrophy.

PURPOSE: Our aim was to analyze retinal structure in young patients with Best disease with reference to future gene therapy. METHODS: This was a retrospective observational spectral domain optical coherence tomography study of four patients aged 10 years or less with Best disease. RESULTS: Findings ranged from subtle thickening at the level of the retinal pigment epithelium-photoreceptor interdigitation line, to subretinal fluid and precipitate-like changes at the level of the photoreceptor outer segments, and further to choroidal neovascularization. The photoreceptor inner segment ellipsoid layer could be visualized seemingly undisturbed above the vitelliform lesions, except in the case of choroidal neovascularization. CONCLUSIONS: Clinical variability is evident even among young patients aged 10 years or less with Best disease. The earliest structural alterations seem to occur at the level of the retinal pigment epithelium-photoreceptor interdigitation line. The photoreceptor inner segment seems to be unaffected unless choroidal neovascularization develops, which seems promising regarding future gene therapy.

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.

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.

Multimodal analysis of the progression of Best vitelliform macular dystrophy.

PURPOSE: To investigate the multimodal morphological features in the different stages of Best vitelliform macular dystrophy (VMD) in subjects harboring mutations in the BEST1 gene, and their changes during the progression of the disease. METHODS: In this retrospective observational study performed between January 2007 and December 2012, 21 patients (42 eyes) with Best VMD from eight families with the BEST1 mutation were included. Best-corrected visual acuity (BCVA), fundus autofluorescence (FAF), and spectral domain optical coherence tomography (SDOCT) were evaluated at study entry and at last visit. RESULTS: The mean age of patients was 26.3±17.4 years. Seven new missense mutations in BEST1 were identified. Mean follow-up was 41.1±18.5 months. Mean BCVA was 0.34±0.34 LogMAR at study entry and 0.32±0.33 LogMAR at last follow-up visit (p = 0.2). The overall lesion area on FAF increased from 6.62±4.9 mm² to 7.34±6.1 mm² (p = 0.05). At study entry, on SD-OCT, photoreceptor inner segment ellipsoid portion (ellipsoid zone, EZ) was normal in 15 eyes, disrupted in 14 eyes, and absent in 13 eyes. In two eyes, EZ changed from normal to disrupted during follow-up. Three eyes of three patients showing pseudohypopyon lesions at study entry progressed to vitelliruptive lesions at the last follow-up visit. Three eyes of three patients showing vitelliruptive lesion at study entry reverted to pseudohypopyon lesion with overall enlargement of the lesion size. CONCLUSIONS: Multimodal analysis allowed documenting a continuous material accumulation and reabsorption in Best VMD progression. Blue FAF and SD-OCT could represent noninvasive imaging techniques to monitor Best VMD.

Screening for BEST1 gene mutations in Chinese patients with bestrophinopathy.

PURPOSE: The purpose of this study was to analyze BEST1 gene mutations in Chinese patients with bestrophinopathy and to describe the clinical features of these patients. METHODS: Thirteen patients from 12 unrelated Chinese families affected by bestrophinopathy were recruited and clinically evaluated with best-corrected visual acuity examination, slit-lamp biomicroscopy, fundus examination and photography, optical coherence tomography, fundus autofluorescence, electro-oculography, and electroretinography. Blood samples were collected for DNA extraction. Mutation analysis was performed by direct sequencing of the BEST1 gene. One hundred control chromosomes were also screened to exclude nonpathogenic polymorphisms. RESULTS: Seven patients showed clinical pictures of Best vitelliform macular dystrophy (BVMD) and harbored heterozygous mutations compatible with autosomal dominant inheritance. Two novel mutations (p.T4I and p.A291V) and three reported mutations (p.R218C, p.Q293H, and p.D301G) were identified. Six patients carried BEST1 mutations on both alleles compatible with autosomal recessive inheritance. Compound heterozygous mutations were detected in four patients who presented a BVMD phenotype, while homozygous mutations were detected in two patients with autosomal recessive bestrophinopathy. Mutation analysis revealed eight mutations. Four (p.Y33H, p.R130L, p.M163R, and c.519delA) were novel, and four (p.R13H, p.A195V, p.R255W, and p.W287*) had previously been reported. CONCLUSIONS: Patients with biallelic BEST1 mutations were common among Chinese patients with bestrophinopathy, and the phenotypes varied. The features and combinations of different BEST1 mutations as well as epistatic effects may influence phenotype expression. Our results expand the BEST1 mutation spectrum.

Frequency and clinical pattern of vitelliform macular dystrophy caused by mutations of interphotoreceptor matrix IMPG1 and IMPG2 genes.

PURPOSE: To assess the frequency of and to characterize the clinical spectrum and optical coherence tomography findings of vitelliform macular dystrophy linked to IMPG1 and IMPG2, 2 new causal genes expressed in the interphotoreceptor matrix. DESIGN: Retrospective epidemiologic, clinical, electrophysiologic, and molecular genetic study. PARTICIPANTS: The database of a national referral center specialized in genetic sensory diseases was screened for patients with a macular vitelliform dystrophy without identified mutation or small deletion or large rearrangement in BEST1 and PRPH2 genes. Forty-nine families were included. METHODS: Clinical, imaging, and electro-oculogram findings were reviewed. Mutation screening of IMPG1 and IMPG2 genes were performed systematically. MAIN OUTCOME MEASURES: Frequency, inheritance, and clinical pattern of vitelliform dystrophy associated with IMPG1 and IMPG2 mutations were characterized. RESULTS: IMPG1 was the causal gene in 3 families (IMPG1 1-3, 11 patients) and IMPG2 in a fourth family (2 patients). With an autosomal dominant transmission, families 1 and 2 had the c.713T→G (p.Leu238Arg) mutation in IMPG1 and family 4 had the c.3230G→T (p.Cys1077Phe) mutation in IMPG2. Patients with IMPG1 or IMPG2 mutations had a late onset and moderate visual impairment (mean visual acuity, 20/40; mean age of onset, 42 years), even in the sporadic case of family 3 with a presumed recessive transmission (age at onset, 38 years; mean visual acuity, 20/50). Drusen-like lesions adjacent to the vitelliform deposits were observed in 9 of 13 patients. The vitelliform material was above the retinal pigment epithelium (RPE) at any stage of the macular dystrophy, and this epithelium was well preserved and maintained its classical reflectivity on spectral-domain optical coherence tomography (SD-OCT). Electro-oculogram results were normal or borderline in 9 cases. CONCLUSIONS: IMPG1 and IMPG2 are new causal genes in 8% of families negative for BEST1 and PRPH2 mutations. These genes should be screened in adult-onset vitelliform dystrophy with (1) moderate visual impairment, (2) drusen-like lesions, (3) normal reflectivity of the RPE line on SD-OCT, and (4) vitelliform deposits located between ellipsoid and interdigitation lines on SD-OCT. These clinical characteristics are not observed in the classical forms of BEST1 or PRPH2 vitelliform dystrophies.

The Retinal Phenotype Associated with the p.Pro101Thr BEST1 Variant.

PURPOSE: To describe the retinal phenotype associated with the p.Pro101Thr BEST1 variant. DESIGN: Retrospective, observational case series. PARTICIPANTS: Patients diagnosed with bestrophinopathies in which molecular genetic testing identified the p.Pro101Thr BEST1 as well as healthy carriers among their first-degree relatives. METHODS: Medical records were reviewed to obtain data on family history and ophthalmic examinations, including retinal imaging. The imaging protocol included OCT and fundus autofluorescence using Spectralis HRA + OCT (Heidelberg Engineering). Genetic analysis was performed by next-generation sequencing. MAIN OUTCOME MEASURES: Results of ophthalmic examinations and multimodal imaging features of retinal phenotypes. RESULTS: The c.301C>A, p.Pro101Thr BEST1 missense variant was identified as the causative variant in 8 individuals (all men) from 5 families, accounting for 13% of cases (8/61) and 10% of pathogenic alleles (9/93) in our cohort of patients affected by bestrophinopathies. Seven individuals (14 eyes) had the variant in heterozygous status: all eyes had a hyperopic refractive error (median spherical equivalent of + 3.75 diopters [D]) and 4 individuals had a macular dystrophy with mildly reduced visual acuity (median of 20/25 Snellen), whereas the other 3 were asymptomatic carriers. On multimodal retinal imaging, 5 (36%) out of 14 eyes had subclinical bestrophinopathy, 4 (29%) had typical findings of adult-onset foveomacular vitelliform dystrophy (AOFVD), and the remaining 5 (36%) displayed a pattern dystrophy-like phenotype. Follow-up data were available for 6 subjects, demonstrating clinical stability up to 11 years, in both subclinical and clinical forms. An additional patient with autosomal recessive bestrophinopathy was found to harbor the p.Pro101Thr variant in homozygosity. CONCLUSIONS: The p.Pro101Thr BEST1 variant is likely a frequent cause of bestrophinopathy in the Italian population and can result in autosomal dominant macular dystrophies with incomplete penetrance and mild clinical manifestations as well as autosomal recessive bestrophinopathy. The spectrum of autosomal dominant maculopathy includes the typical AOFVD and a pattern dystrophy-like phenotype. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Typical best vitelliform dystrophy secondary to biallelic variants in BEST1.

BACKGROUND: Pathogenic variants in BEST1 can cause autosomal dominant or autosomal recessive dystrophy, typically associated with distinct retinal phenotypes. In heterozygous cases, the disorder is commonly characterized by yellow sub-macular lesions in the early stages, known as Best vitelliform macular dystrophy (BVMD). Biallelic variants usually cause a more severe phenotype including diffuse retinal pigment epithelial irregularity and widespread generalized progressive retinopathy, known as autosomal recessive bestrophinopathy (ARB). This study describes three cases with clinical changes consistent with BVMD, however, unusually associated with autosomal recessive inheritance. MATERIALS AND METHODS: Detailed ophthalmic workup included comprehensive ophthalmologic examination, multimodal retinal imaging, full-field and pattern electroretinography (ERG; PERG), and electrooculogram (EOG). Genetic analysis of probands and segregation testing and fundus examination of proband relatives was performed where possible. RESULTS: Three unrelated cases presented with a clinical phenotype typical for BVMD and were found to have biallelic disease-causing variants in BEST1. PERG P50 and ERG were normal in all cases. The EOG was subnormal (probands 1 and 3) or normal/borderline (proband 2). Probands 1 and 2 were homozygous for the BEST1 missense variant c.139C>T, p.Arg47Cys, while proband 3 was homozygous for a deletion, c.536_538delACA, p.Asn179del. The parents of proband 1 were phenotypically normal. Parents of proband 1 and 2 were heterozygous for the same missense variant. CONCLUSIONS: Individuals with biallelic variants in BEST1 can present with a phenotype indistinguishable from BVMD. The same clinical phenotype may not be evident in those harboring the same variants in the heterozygous state. This has implications for genetic counselling and prognosticationA.