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.

Introduction BEST1 (VMD2) is a gene located on chromosome 11 (11q12.3) that encodes for the 585 amino acid transmembrane protein bestrophin 1 which is located on the basolateral aspect of retinal pigment epithelial (RPE) cells [1,2]. Although the functional role of bestrophin-1 within the RPE has not been determined definitively, it has been postulated to function as a Ca 2? -activated Clchannel [3], a regulator of voltagegated Ca 2? channels [4], and a HCO 3 channel [5]. Mutations in BEST1 therefore affect the RPE metabolism and consequently the outer retinal function with which the RPE is intimately associated.
Autosomal recessive bestrophinopathy (ARB), first described in detail in 2008, is another member of the phenotypic spectrum associated with mutations in the BEST1 gene [11]. The characteristics of this disorder include a progressive reduction in central vision, absence of the electro-oculographic (EOG) light rise, and reduced full-field electroretinograms (ERGs). None of the patients have the vitelliform lesions typical of Best disease, but have a diffuse irregularity of the reflex from the RPE including dispersed punctate flecks [11]. All of the patients have an accumulation of fluid within and/or beneath the neurosensory retina in the macular area [11].
ARB has been reported to be due to either compound heterozygous or homozygous BEST1 gene mutations [6,11]. A recent manuscript described an ocular phenotype similar to ARB associated with a single heterozygous mutation of the BEST1 gene [12].
Several mutations associated with ARB have been reported to be involved in causing dominant Best disease when they were present in the heterozygous state [6,7,[13][14][15]. The clinical phenotype of some patients with recessive bestrophinopathy is distinct from that seen in Best disease, while in others it is similar to the typical phenotype observed in autosomal dominant vitelliform dystrophy [16,17]. Identification of additional families with recessive bestrophinopathy and detailed characterization of the clinical phenotypes of homozygous and heterozygous individuals will assist in establishing the phenotype-genotype correlations in patients with BEST1-associated diseases.

Methods
The protocol of this study conformed to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the Nippon Medical School. A signed written informed consent was obtained from the patient and his parents after the nature and possible consequences of the study were explained.
Blood samples were collected from the patient and his parents, and genomic DNA was isolated from the peripheral white blood cells using a blood DNA isolation kit (NucleoSpin Blood XL; Macherey-Nagel, Germany). The DNA was used as the template to amplify the BEST1 gene. The coding regions and flanking introns of the BEST1 gene were amplified by polymerase chain reaction (PCR) using primers synthesized by Greiner Bio-One (Tokyo, Japan). The PCR products were purified (ExoSAP-IT; USB Corp., USA) and were used as the template for sequencing. Both strands were sequenced on an automated sequencer (Bio Matrix Research; Chiba, Japan).
The ophthalmological examinations included measurements of the best-corrected visual acuity (BCVA), refractive error and axial length, slit-lamp biomicroscopy, ophthalmoscopy, fundus photography, fundus autofluorescence (FAF) imaging, fluorescein angiography (FA), SD-OCT, full-field electroretinography (ERG), multifocal ERGs (mfERGs), and electro-oculography (EOG). The EOGs and ERGs were recorded using an extended testing protocol conforming to the International Society for Clinical Electrophysiology of Vision standards. The ERGs were elicited and recorded with a LED built-in electrode (LE2000, Tomey, Japan). The mfERGs were recorded using a commercial mfERG system (VERIS Science; Electro-Diagnostic Imaging, Inc. Redwood City, CA, USA). The FAF images were acquired with the TRC-NW8Fplus retinal camera (TOPCON, Tokyo, Japan),

Results
The patient was a 25-year-old man whose decimal best-corrected visual acuity (BCVA) was 0.9 in the   (Fig. 1). The vitelliform lesions that are typical of Best disease were not observed (Fig. 1). FAF imaging showed multiple hyper-autofluorescent spots in the peripheral retina of both eyes, and the site of the spots corresponded with the yellowish deposits observed by ophthalmoscopy (Fig. 1). FAF imaging also detected a hypoautofluorescent lesion in the macula of both eyes (Fig. 1). FA showed widespread patchy hyper-fluorescence (Fig. 1). The SD-OCT images showed cystoid changes in the macula and shallow serous retinal detachments in both eyes. There was a thickening and hyper-reflectivity at the areas corresponding to ellipsoid and interdigitation zones of the photoreceptors in the SD-OCT images (Fig. 1). The amplitudes of both the cone and rod full-field ERGs were reduced, and the waveforms were similar in both eyes (Fig. 2). The amplitudes of the mfERGs were reduced in the central and peripheral sectors of both eyes (Fig. 3). The Arden ratio of the EOG was 1.1 in both eyes with a dark trough 15 min after beginning the measurements and a light peak 15 min from the beginning of the light phase (Fig. 4).
Mutation analysis of the BEST1 gene in the proband showed two heterozygous sequence variants. One was a novel variant, c.717delG, p.V239VfsX2, and the other was a variant previously reported, c.763C[T, p.R255W. Both variants were found in exon 7 (Fig. 5).
The proband's father (I-1, 57 years old) and mother (I-2, 57 years old) had normal visual acuity, and their fundus, FAF, and SD-OCT images were also within normal limits (Fig. 6). The EOGs of both parents had a normal light rise with normal Arden ratio in both eyes (Fig. 5). Mutation analyses of the parents identified a c.717delG, p.V239VfsX2 variant in the father and a c.763C[T, p.R255W variant in the mother in the heterozygous state.

Discussion
The imaging and functional data obtained on our patient are in good agreement with the findings from previous reports of ARB. The characteristic features of ARB are a clinically recognizable retinal dystrophy with yellowish subretinal lesions scattered in the posterior pole that have marked diffuse fundus autofluorescence abnormalities [18][19][20][21][22][23][24]. The SD-OCT findings of previous ARB cases included diffuse intraretinal cystic spaces across both the inner and   BVMD Sodi et al. [33] outer plexiform layers, subretinal fluid with shallow serous retinal detachment, and thickening and hyperreflectivity of the ellipsoid and interdigitation zones which may represent an elongation of the photoreceptors [21,24,25]. The Arden ratio of the EOGs of patients with ARB is reported to be low with an absence of the light rise [11,19,23]. The imaging and functional findings in our patient are typical of ARB. The BEST1 mutation, c.717delG, p.V239VfsX2, has not been reported and not included in the SNP database. The allele frequency of the variant was estimated from two databases; the Human Genetic Variation Database (HGVD; http://www.genome. med.kyoto-u.ac.jp/SnpDB/about.htm) which is specific for the Japanese population, and the ExAC Browser (Beta)(http://exac.broadinstitute.org) database. Both of these databases did not contain the allele frequency of the variant, which indicates that this variant is very rare.
Although most mutations associated with BVMD are missense mutations that do not compromise protein synthesis, the few ARB-causing mutations reported to date are premature truncations or nonsense substitutions that lead to early transcript degradation or non-functional proteins. These are associated with a null phenotype (Table 1). In truncating BEST1 mutations, the null phenotype associated with ARB is attributed to a severe decrease in BEST1 expression promoted by the nonsense-mediated decay (NMD) surveillance mechanism [26]. Recent evidence supports the idea that NMD degradation depends on the position of the premature translation termination codons. Pomares et al. [26] reported that the BEST1 transcripts in a patient who carried the premature stop codon at position 230 are preserved in only 13 % of the case, while the BEST1 transcripts of a patient who carry a premature stop codon in position 349 are preserved in 22 % of the case. Patients who carry the premature stop codon in position 230 have a characteristic ARB phenotype, while patients who carry a premature stop codon in position 349 have ophthalmological features resembling both ARB and BVMD [26]. Thus, the residual amount of aberrant protein can promote a negative effect causing a mixed phenotype of both ARB and BVMD traits. This hypothesis was supported by previous reports of biallelic BEST1 mutations with at least a premature termination codon (Table 1). Although patients 2 and 6 of Table 1 had the BVMD phenotype which is not consistent with the hypothesis, the same second allele mutation (R141H) may be associated with the BVMD phenotype [21,27]. Our patient with a premature termination codon at position 240 is consistent with the hypothesis that the patient should have an ARB phenotype.
The other mutation found in this study (R255W) was reported to be present in both a BVMD family in the heterozygous state and two ARB families in the compound heterozygous state [28,29]. In the BVMD family with the R255W mutation, the parents of the proband were not genetically examined [28]. In the ARB families with the R255W mutation, each parent of the proband was heterozygous carriers of the R255W mutation and they were healthy [29]. Our data do not explain why the mother of our patient who carried the heterozygous R255W mutation did not have BVMD. One possibility is that the mutation exhibits reduced penetrance for the phenotype. The other possibility is that the previously described BVMD patient who had heterozygous c.763C[T, p.R255W mutation may have had an undiscovered second allele mutation such as a large deletion.
In some cases, it is difficult to differentiate ARB from BVMD and to speculate on the prognosis of the disease. Identifying the genetic defect of BEST1 gene and position of the premature termination codon may help in differentiating the ARB from BVMD and predict the prognosis of the disease.
Statement of welfare of animals No animals were involved in the study.
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