Keywords

21.1 Introduction

Macular dystrophy is a group of heritable disorders that cause ophthalmoscopically visible macular abnormalities. Vitelliform macular dystrophy (VMD) is a group of macular dystrophy characterized by the subretinal accumulation of yellow yolk-like materials which predominantly affects the macula. Best vitelliform macular dystrophy (BVMD) is named after Friedrich Best who described a family with a history of early-onset macular degeneration in 1905 [1]. BVMD is among the most common autosomal dominant (AD) retinal dystrophy, caused by mutations in the BEST1 gene . Since the first identification of BEST1 gene in 1998 [2], molecular biology and pathophysiology of BEST1 gene in VMD have been studied. Recent advances in genetic analysis have described over 200 different human BEST1 mutations to date, associated with a broad spectrum of ocular diseases , called a bestrophinopathy [3, 4]. Bestrophinopathy includes five clinically distinct categories BVMD, adult-onset vitelliform macular dystrophy (AVMD) , autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa . AVMD was first described by Gass in 1974 who initially termed it peculiar foveomacular dystrophy [5]. AVMD is one of the most common forms of macular dystrophy as well [6]. Many investigators suggested that AVMD is a mild form of BVMD within the same spectrum because the clinical features of AVMD were similar to those of early-stage BVMD and the age of onset were highly variable [7,8,9]. Clinically, BVMD is distinguished from AVMD by earlier age of onset, larger lesion size, and an abnormal electrooculogram (EOG). Clinical features of VMD are similar to those of exudative age-related macular degeneration (AMD) , choroidal neovascularization (CNV), or central serous chorioretinopathy (CSC). Thus, genetic test is clinically important in the diagnosis of VMD. Here, in addition to describing the clinical characteristics of VMD, this chapter focuses on the clinical genetics of BEST1 gene in VMD (BVMD and AVMD).

21.2 Epidemiology and Asian Perspective

VMD is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 10,000 in the USA [10], 2/10,000 in Sweden [11], 1.5/100,000 in Denmark [12], and 1 in 16,500 to 1 in 21,000 in Olmsted County, Minnesota, USA [13]. Males are more affected than females (3:1) [11, 12]. Despite the update of novel mutations of BEST1 in Asian VMD patients, there was no report of the prevalence of VMD in Asian countries. Thus, a study of the prevalence of VMD with a genetic analysis in Asian countries is necessary.

21.3 Molecular Biology

The BEST1 gene consists of 11 exons that encode the bestrophin-1 protein (585 amino acids). Bestrophin-1 is a retinal pigment epithelium (RPE) protein hypothesized to function as a Ca2+-activated Cl- channel (CaCC), or a regulator of ion transport [14]. Bestrophin-1 is predominantly expressed in the basolateral membrane of the RPE [15]. X-ray structure of chicken BEST1-Fab complexes indicates that Bestrophin-1 forms a homo-pentamer and functions as a CaCC [16]. Disease-causing mutations are prevalent within the gating apparatus. In addition, Bestrophin-1 functions as a regulator of intracellular calcium signaling and influences transepithelial electrical properties [17]. Recently, patient’s stem cell-derived RPE is used for the function of bestrophin-1 and reveals that bestrophin-1 assembles into a key calcium-sensing chloride channel in human RPE [18]. Further study using RPE cells from patient-derived induced pluripotent stem cells (iPSc) harboring BEST1 mutations is required to elucidate the exact functional role of bestrophin-1.

21.4 Clinical Features

21.4.1 BVMD

BVMD is an early-onset autosomal dominant disorder showing extremely variable penetrance and expressivity. The diagnosis of BVMD shows a bimodal age distribution; the first maximum peak was made during the childhood, but the second peak was made following puberty and extending into the sixth decade of life [19]. Before the era of genetic analysis, the diagnosis of BVMD was based on typical fundus findings, family history, and a decreased Arden ratio (light peak/dark trough) of EOG with a normal electroretinogram (ERG), which may contribute to variability of penetrance, expressivity, and onset age.

BVMD is caused by dysfunction of Bestrophin-1 protein, a CaCC protein located on the basolateral membrane of RPE, which causes abnormal fluid and ion exchange that decreases pumping of fluid from the subretinal space, and results in swelling of RPE and subretinal lipofuscin accumulation [20]. Histopathologically, autofluorescent material was accumulated in the outer retina and the subretinal space in BVMD, which is considered as indigestible components of photoreceptor outer segments that accumulate due to the lack of direct apposition of the outer segments and the RPE [21]. Eventual phagocytosis of these older materials over time would load the RPE cells and may account for excessive accumulation of abnormal lipofuscin in RPE cells across the entire fundus [22]. These findings coincide with the decreased Arden ratio of EOG, less than 1.5, seen in BVMD, which suggest generalized dysfunction of the RPE. Even otherwise asymptomatic carriers of BEST1 mutations will exhibit an altered EOG [23]. Full-field ERG is generally normal, but the multifocal ERG amplitudes of the central and pericentral responses were significantly reduced in the majority of patients [24]. However, the photoreceptor structure evaluated cellular imaging with adaptive optics scanning light ophthalmoscopy was retained within active BVMD lesions, even in apparently advanced disease [25, 26].

Five progressive stages can be defined based on fundus examination [20, 27]. However, these stages are not observed in all patients, nor do they occur consecutively. The first previtelliform stage is characterized by the absence of symptoms and subtle RPE changes such as RPE mottling and a small yellow spot. On optical coherence tomography (OCT), RPE and ellipsoid zone (EZ) disruption was detectable in a small fraction of eyes [28, 29]. A slight thickening of the interdigitation zone was also observed [30]. EOG is abnormal and fluorescein angiogram (FA) shows window defects. Visual acuity remains intact in most patients. The previtelliform lesions are characterized by absence or only slight autofluorescence on fundus autofluorescence (FAF) imaging.

The second vitelliform shows a well-circumscribed, circular, homogeneous, yellow-opaque, 0.5–3 disc diameter sized, yolk-like macular lesions. The remaining part of fundus usually has normal appearance, but multifocal lesions also can be seen. The accumulation of hyperreflective vitelliform material is clearly visible on OCT below the neurosensory retina , located between the EZ and the RPE. The disruption of outer retinal layers and neurosensory retinal detachment with subretinal fluid occur in many cases [28, 29]. The yellowish subretinal material is intensely hyperautofluorescent in FAF imaging. FA shows marked hypofluorescence in the zone covered by lesion by blockage of fluorescence. Metamorphopsia, blurred vision, and a decrease of central vision can occur.

In the third pseudohypopyon stage, the vitelliform material accumulates inferiorly and develops a fluid level. On OCT, the upper part of the lesion is observed as hyporeflective area located between RPE and EZ, with clumping of hyperreflective material on the posterior retinal surface. The lower part of the lesion, where the vitelliform material is still accumulated, shows a highly reflective area located in the subretinal space. FA shows hypofluorescence in the lower part resulted from the blockage by the vitelline material. The superior part shows hyperfluorescent due to transmission defects linked to RPE and chorioretinal atrophy in the early phase. FAF shows loss of autofluorescence, particularly in the upper part.

The fourth vitelliruptive stage is characterized by the partial reabsorption of the vitelliform material. This vitelliform material becomes less homogeneous to develop a “scrambled-egg” appearance. OCT shows an optically empty lesion between EZ and RPE, with clumping of hyperreflective material on the posterior retinal surface like the upper part of the pseudohypopyon. The areas of focal RPE hypertrophy can be observed as hyperreflective mottling on the RPE layer on some parts. FAF shows decreased of autofluorescence centrally but increased autofluorescence at the outer border of the lesion.

In the last atrophic/fibrotic stage, RPE atrophy and loss of central vision occur after rupture and reabsorption of the cystic lesion. FA shows hyperfluorescence without leakage. OCT reveals thinning of all the retinal layers and diffuse disappearance of outer retinal layers within the macular area, with highly hyperreflective thickening at the RPE level [29, 31]. Atrophic lesions are characterized by decreased autofluorescence on FAF.

Choroidal neovascularization (CNV) may develop and can lead to form a disciform scar. Patients usually underwent sudden visual disturbance with central scotoma and/or metamorphopsia, showing a macular hemorrhage on fundus examination. In that case, FA shows hyperfluorescence because of CNV and leakage. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agent was effective in treating CNV complicated with BVMD and safe even in children [32,33,34].

Patients with BVMD undergo a progressive decrease of vision over time. In a study that evaluated the course of visual decline of 53 patients in BVMD with BEST1 mutation [35], the median age of onset of visual symptoms was 33 years. Twenty-five percent of patients retain visual acuity of 20/40 or better at the age of 66 years. Other study evaluated 47 patient with BVMD; 74% of patient older than 30 years had 20/100 or worse visual acuity at least one eye [36].

21.4.2 AVMD

Gass reported a three-generation family and six sporadic patients characterized by one-third disc diameter sized bilateral subfoveal vitelliform lesions with onset between the ages of 30 and 50 years accompanied by slowly progressive visual loss as “peculiar foveomacular dystrophy.” They also showed occasional paracentral drusen, normal to slightly subnormal response on EOG but normal ERG and color vision [5]. AVMD shows a variable genetic inheritance, although most cases are sporadic [37]. Patients with AVMD may be asymptomatic but become symptomatic in the fourth or fifth decade of life with blurred vision, metamorphopsia, or scotoma and typically have slow progression of vision loss [38]. Patients with AVMD typically present a round, yellowish subretinal deposit in one-third to one disc diameter size within the macular area, similar fundus finding to the vitelliform stage of BVMD.

The initial yellow lesion may present in only one eye and appear as small yellow flecks in the paracentral area. EOG shows a normal or slightly reduced Arden ratio, which is obviously abnormal in BVMD. The macular lesion appears as hyperautofluorescent in FAF. The vitelliform deposit usually appears as initially hypofluorescent but gradually becomes hyperfluorescent on the edges by staining of the dye in FA [39] and hypofluorescent on indocyanine green angiography (ICGA). OCT reveals a dome-shaped hyperreflective lesion located between the retina and RPE [40]. The foveal thinning and EZ disruption are also observed and probably explain the progressive visual loss [41, 42].

AVMD progression is characterized by fragmentation and reabsorption of the vitelliform material [6]. Macular atrophy progressively replaces the vitelliform deposits at the advanced stages of the disease in most cases [42], but most patients retain reading vision throughout life [43, 44]. CNV may be complicated in few cases; 6 out of 51 patients developed CNV after a 6-year follow-up [45]. Anti-VEGF therapies have shown to be effective in the treatment of CNV associated with AVMD [46].

21.5 Genetic Aspects

21.5.1 BVMD

Currently, only genetic test for mutation analysis of the BEST1 gene leads to confirmation of a clinical diagnosis of BVMD. Note that individuals with clinical findings of BVMD occasionally have a normal EOG, turning out to have a pathogenic variant of BEST1 [47]. In case of atypical BVMD [3], genetic test for confirmation should be performed. Over 200 BEST1 mutations with significant clinical heterogeneity require a thorough genetic analysis and clinical examinations to better understanding of genotype -phenotype correlations in BVMD. Most mutations of BEST1 gene in BVMD and AVMD are missense mutations. Table 21.1 shows a list of missense mutations of BEST1 gene in BVMD and AVMD.

Table 21.1 BEST1 missense mutations in BVMD and AVMD
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Most genetic studies were performed in Western countries including the USA, England, Sweden, Denmark, Germany, the Netherlands, Italy, and France. BEST1 mutations are extremely heterogenous, but several mutations have been frequently found (Thr6Pro, Arg25Trp, Arg218Cys, Tyr227Asn, Arg243Val, Ile295del, Gle300Asp, Asp301Glu, and Asp302Asn). Interestingly, these frequent mutations are ethnic specific (44.4% of Asp302Asn in Danish [12] and 36.8% of Arg25Trp in Italian [86]).

Currently, only limited reports are available in Asian genetic studies of BEST1 from Chinese [50, 54, 70, 76, 80, 87,88,89], Japanese [48, 83], and Korean [9]. The mutation spectrum of the BEST1 gene in Asian patients of BVMD is differed from those in Western patients [88]. Six novel missense mutations (Thr2Asn, Leu75Phe, Ser144Asn, Arg255Trp, Pro297Thr, and Asp301Gly) and one previously reported mutation (Arg218Cys) were identified [50]. Three novel mutations Tyr4Ile [54], Ala291Val [54], and Phe113Leu [76] in BVMD were reported. Lin [80] reported two novel heterozygous mutations 304delAsp and Trp229Gly in Chinese BVMD patients. Liu [70] reported four previously reported mutations (Ser16Phe, Ser144Asn, Glu292Lys, and Glu300Lys) and two novel disease-causing mutations (Thr307Asp, Arg47His) in Chinese patients with BVMD.

In Japanese study [48], 22 patients including 16 probands from 16 families with BVMD were analyzed. All 16 probands exhibited characteristic BVMD fundus appearances, abnormal EOG, and normal ERG responses with the exception of one diabetic retinopathy proband. Genetic analysis identified 12 BEST1 variants in 13 probands (81%). Of these, ten variants (Tyr2Arg, Arg25Trp, Phe80Leu, Val81Met, Ala195Val, Arg218His, Gly222Glu, Val242Met, Asp304del, and Glu306Asp) have been previously reported in BVMD, while two variants (Ser7Asn and Pro346His) were novel disease-causing mutations.

In Korea, we report a BVMD patient (Fig. 21.1) carrying Asn296Lys mutation which is a causative mutation of multifocal BVMD in German patient [60]. Arg218Leu is a novel disease-causing mutation in BVMD (Fig. 21.2). These findings expand the spectrum of BEST1 genetic variation in Asian and will be valuable for genetic counseling for patients with BVMD [88].

Fig. 21.1
figure 1

Best vitelliform macular dystrophy (BVMD). A 32-year-old man carrying p.Asn296Lys mutation in the BEST1 gene was incidentally found on routine fundus examination for a pilot license. The visual acuities (VA) were 20/20 in both eyes. (a, f) Bilateral BVMD of vitelliruptive stage shows scattered yellow-white vitelliform deposits. (b, g) Vertical optical coherent tomography (OCT) shows serous retinal detachment and hyperreflective vitelliform materials at RPE in both eyes. (c, h) Fluorescein angiography (FA) shows late pooling of fluorescein dye at the egg lesion. (d, i) Fundus autofluorescence (FAF) image of the vitelliruptive lesion shows increased autofluorescence at inferior part of ruptured vitelliform lesions and at the border of the serous retinal detachment. (e, j) Indocyanine green angiography (ICGA) shows active leakage spot in the left eye

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Fig. 21.2
figure 2

Best vitelliform macular dystrophy (BVMD). A 39-year-old man carrying Arg218Leu mutation in the BEST1 gene had multiple injections of anti-VEGF agents (ten for right eye and five for left eye) in both eyes. At initial visiting in our institute, vitelliform stage of right eye (a) reveals highly reflective subfoveal pillar without surrounding SRF (b). Small round vitelliform lesion with central cicatricial change was found in the left eye (e). OCT reveals marked RPE loss at the fovea (f). Six months later, FAF shows dispersed materials with hyperautofluorescent (c), and OCT reveals the disappearance of subfoveal pillar with a progression to vitelliruptive stage (d) in the right eye. FAF shows central hypoautofluorescent and surrounding hyperfluorescent lesions. OCT reveals that hypoautofluorescent lesion corresponds to the enlarged RPE loss (h)

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BVMD shows variable expressivity and incomplete penetrance at the clinical level. Disease-causing effect of BEST1 mutations seems to be cumulative over time [79]. In genotype -phenotype relationship of Dutch study [59], median age of onset of visual symptoms was 33 years (range, 2–78). The cumulative risk of VA below 0.5 (20/40) was 50% at 55 years and 75% at 66 years. The cumulative risk of VA decline less than 0.3 (20/63) was 50% by age 66 years and 75% by age 74 years. Most patients (96%) had missense mutations; the Thr6Pro, Ala10Val, and Tyr227Asn mutations were most common. Visual decline was significantly faster in patients with an Ala10Val mutation than either the Thr6Pro or the Tyr227Asn mutation.

In the recent Chinese study, despite typical macular appearance of BVMD, no clear genotype-phenotype correlation was observed [88]. In Asian BVMD cohort, genetic tests should be performed for the diagnosis with thorough clinical examinations to elucidate a genotype -phenotype correlation.

21.5.2 AVMD

In AVMD, several mutations in BEST1 gene have been identified including p.Ala146Lys [90], p.Thr6Pro, p.Arg47His, P.Ala243Val, p.Asp312Asn [61], and p.Ile38Ser [9]. Table 21.1 includes the list of missense mutations in AVMD. In addition, AVMD is associated with mutations in PRPH2 [91], IMPG1 [92], IMPG2 [93].

Age of onset is a major criterion to distinguish BVMD from AVMD [64]. Thus, systematic screening of BEST1 and PRPH2 has been suggested in BVMD and AVMD. BEST1 screening should be recommended to patients with an age of onset less than 40 years, and PRPH2 screening should be recommended to patients with an age of onset more than 40 years. For an onset between 30 and 40 years, PRPH2 can be screened if no mutation has been detected in BEST1. In this screening approach, we found PRPH2 mutation of p.Pro219_Pro221delinsPro in a 39-year-old female without BEST1 mutation (Fig. 21.3).

Fig. 21.3
figure 3

Adult-onset vitelliform macular dystrophy (AVMD) . A 39-year-old woman carrying Pro219_Pro221delinsPro in PRPH2 gene suffered from dysmorphopsia of the right eye. OCT reveals subfoveal vitelliform lesion in the right eye (a) and left eye (b)

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21.6 Future Perspectives for Therapy

The development of gene and cell therapies is promising in various retinal diseases . Indeed, the results of clinical trials using iPSC-derived RPE cells in wet age-related macular degeneration [94] or AAV/RPE65 vectors in Leber’s congenital amaurosis [95] were already reported. Therapeutic intervention of inherited retinal dystrophy should be primarily aimed at the restoration of normal gene (i.e., BEST1 gene in BVMD and AVMD) . However, until decade ago, this therapeutic goal was ideal but unachievable due to the lack of a proper biotechnology. Recent advances in genome editing technology using CRISPR system and gene delivery system are promising and harness the CRISPR-based genome editing for the therapeutic applications. Since its first therapeutic applications in retinal disease using wet AMD animal models [96, 97], in vivo genome editing using CRISPR-Cas9 enlarged its therapeutic applications both in genetic diseases harboring mutations [98, 99] and nongenetic degenerative diseases [96, 97, 100].

Conventional concept of gene therapy to deliver normal copy of BEST1 gene into RPE would be effective in the treatment of VMD of haploinsufficiency phenotype , which is caused by BEST1 mutations that exclusively result in a loss of sufficient wild-type protein. In addition, simple destruction of mutant proteins at the DNA level is achievable by genome editing of mutant BEST1 allele using CRISPR-Cas9.

Currently, many BEST1 mutations cause VMD through dominant negative effect. In addition, over 200 mutations of BEST1 gene, large amounts of BEST1 mutations are missense mutations; thus, a precise base-editing using base-editors enables a literally complete recovery of normal gene [101, 102]. According to the recent advances in genome editing technology using CRISPR system, in vivo genome editing has emerged as a potential treatment strategy for inherited retinal dystrophies [103].

21.7 Summary

VMD is among the most common autosomal dominant macular dystrophy. Multimodal imaging with SD-OCT, FAF, FA, and ICGA is useful to the diagnosis of VMD. Genetic test is clinically important in the diagnosis of VMD because the clinical features of VMD can be similar to those of exudative AMD , CNV, or CSC. Future studies are needed to identify the prevalence with precise genetic mutations of BEST1 in Asian VMD patients. This could provide a clear genotype -phenotype correlation in VMD. In vitro studies using RPE cells from patient-derived iPSC help to understand molecular biology of bestrophin-1 protein. Furthermore, in vivo genome editing using CRISPR-based base-editors might be a potential treatment strategy for the correction of missense mutations in VMD.