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.

Introduction

The human BEST1 gene (OMIM 607854; previously known as VMD2) was mapped on the long arm of chromosome 11q12-q13 and found to be causative for Best vitelliform macular dystrophy (BVMD) in 1998 by linkage and sequencing studies of families affected by BVMD [1]. The gene consists of 11 exons that encode a 585-amino acid transmembrane protein, bestrophin-1, which localizes to the basolateral membrane of RPE cells [2]. To date, more than 200 different BEST1 mutations have been identified, most of which are missense mutations located in the N-terminal half of the protein (HGMD). These mutations are associated with at least four clinically distinguishable degenerative human eye diseases, collectively referred to as bestrophinopathies: BVMD or Best disease (OMIM 153700), autosomal recessive (ar) bestrophinopathy (ARB, OMIM 611809), autosomal dominant (ad) vitreoretinochoroidopathy (OMIM 193220), and adult-onset vitelliform macular degeneration (OMIM 608161) [3]. BEST1 mutations have also been implicated in retinitis pigmentosa (RP) and microcornea, retinal dystrophy, cataract, and posterior staphyloma (MRCS) syndrome in rare cases [4,5].

BVMD, initially described in 1905 by the German ophthalmologist Friedrich Best [6], is by far the most common disease associated with heterozygous BEST1 mutations. It is characterized by a yellowish yolk-like lesion in the macula and a markedly abnormal electro-oculogram (EOG) with a reduced light-to-peak ratio (Arden ratio) that is less than the cutoff value of 1.5 [6-9]. According to Gass, the macular lesion evolves through various well-defined stages with time: vitelliform, pseudohypopyon, vitelliruptive (scrambled egg), atrophic, and cicatricial [10]. Some patients may develop secondary choroidal neovascularization that could lead to severe visual loss [10]. BVMD is usually inherited as an AD trait with incomplete penetrance and considerable variability in phenotypic expression [11], albeit a few families with AR inheritance have also been reported [12-15]. Studies on related BEST1 mutations suggested that the pathologic mechanism could involve a dominant negative effect, haploinsufficiency, or total loss of function depending on the nature of the protein change [16].

Another distinct retinal disorder, ARB, was first reported by Burgess et al. [16]. The most common distinguishing features of ARB are extrafoveal and extramacular subretinal deposits and an accumulation of fluid within and/or beneath the neurosensory retina in the macula without vitelliform lesions typical of BVMD. In contrast to most BVMD cases, full-field electroretinography (ERG) shows reduced and delayed rod and cone responses and severe reduction or absence of the EOG light rise. ARB has been found in association with either homozygous or compound heterozygous BEST1 mutations [16].

To date, most genetic and phenotypic studies of bestrophinopathy have been performed in Western populations, and only limited data are available for Chinese patients [17-19]. The aim of the present study was to screen for BEST1 gene mutations in Chinese patients affected by bestrophinopathy and to describe their clinical features.

Methods

This study was approved by the Institutional Review Board of Peking Union Medical College Hospital. Informed written consent in accordance with the tenets of the Declaration of Helsinki and the Guidance of Sample Collection of Human Genetic Diseases by the Ministry of Public Health of China was obtained from the participating individuals or their guardians before the participants enrolled in study.

Clinical studies

Thirteen patients diagnosed with bestrophinopathy from 12 independent pedigrees were recruited from the Department of Ophthalmology, Peking Union Medical College Hospital (Figure 1). Detailed ophthalmic examinations were conducted on all subjects, including best-corrected visual acuity (BCVA), slit-lamp biomicroscopy, fundus examination and photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), EOG, and ERG. The fundus images and OCT images were in most cases taken with Topcon-3D OCT-1000 (Topcon Medical Systems, Tokyo, Japan), and FAF examinations were performed with the Spectralis HRA-OCT produced by Heidelberg Engineering (Heidelberg, Germany). EOG and ERG (RetiPort ERG system; Roland Consult, Wiesbaden, Germany) were performed according to the International Society for Clinical Electrophysiology of Vision (ISCEV) standards [20,21].

Figure 1

Pedigrees of the 12 Chinese families included in this study and segregation analysis of the biallelic mutations of the BEST1 gene. Squares represent men, and circles represent women. Solid symbols indicate patients affected with bestrophinopathy. Unfilled symbols represent unaffected family members. A diagonal line indicates a deceased family member. The arrow indicates the proband.

Molecular genetic studies

Peripheral blood samples were obtained from all subjects, and genomic DNA was extracted by using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). All exons and the flanking introns of the BEST1 gene were amplified with PCR using previously reported primers [22] and directly sequenced on an ABI 3730 Genetic Analyzer (ABI, Foster City, CA). PCR conditions were 94 °C 5 min; 30 cycles of 94 °C, 30 s, exon-specific annealing temperature TA, 30 s, 72 °C, 30 s; 72 °C, 5 min. The results were analyzed with Laser-gene SeqMan software (DNASTAR, Madison, WI) and compared with a BEST1 reference sequence (GeneBank accession number NM_004183). One hundred control chromosomes from the same ethnic background were also screened to exclude nonpathogenic polymorphisms.

Results

Clinical evaluation

All patients from families A–F had the typical fundus appearance of BVMD, ranging from vitelliform lesion to pseudohypopyon change. Typical Arden ratios of EOG less than the cutoff value of 1.5 were observed. Age at onset of the disease varied widely in these patients (mean age ± standard deviation [SD] 25.9±16.6 years), as was visual acuity. BCVA ranged from 20/100 to 20/32. Detailed clinical data of these patients are summarized in Table 1. Fundus photographs, OCT, and FAF images of selected patients are shown in Figure 2.

Table 1

Clinical features of patients with mutations in the BEST1 gene

Family	Patient	Age/ Gender	Age at onset	BCVA (R,L)	Fundus finding	EOG Arden ratio	ERG	Genotype
A	II:1	32Y/M	21Y	20/40 20/32	OD pseudohypopyon OS vitelliruptive	NA	NA	p.Q293H/+
B	II:1	6Y/M	4Y	20/40 20/50	OU vitelliform lesion	NA	NA	p.R218C/+
C	I:1	36Y/M	30Y	20/50 20/50	OU pseudohypopyon	OD 1.32 OS 1.25	Normal	p.T4I/+
C	II:1	10Y/F	9Y	20/40 20/40	OD pseudohypopyon OS vitelliruptive	OD 1.28 OS 1.30	Normal	p.T4I/+
D	II:1	45Y/M	42Y	20/40 20/32	OD pseudohypopyon OS vitelliform lesion	OD 1.34 OS 1.21	Normal	p.D301G/+
E	II:1	58Y/F	50Y	20/32 20/100	OU pseudohypopyon	NA	NA	p.A291V/+
F	III:1	26Y/ M	25Y	20/32 20/40	OU pseudohypopyon	NA	NA	p.R218C/+
G	II:1	6Y/M	5Y	20/200 20/63	OD subretinal fibrosisi OS subretinal fibrosisi; yellowish deposits	OD 0.95 OS 1.18	Reduced scotopic and photopic responses	p.M163R/p.M163R
H	II:1	26Y/M	23Y	20/63 20/200	OU yellowish deposits	OD 1.11 OS 1.23	Reduced scotopic and photopic responses	p.R130L/p.R130L
I	II:1	11Y/M	8Y	20/32 20/32	OU macular scar	NA	NA	c.519delA/p.W287*
J	II:2	11Y/F	9Y	20/200 20/40	OD macular scar OS vitelliruptive	NA	NA	p.Y33H/p.R255W
K	II:2	15Y/M	11Y	20/63 20/400	OU hyperpigmented scar	OD 1.20 OS 1.32	Normal	p.R13H/p.A195V
L	II:1	25Y	22Y	20/50 20/40	OD pseudohypopyon OS vitelliform lesion	NA	NA	p.A195V/p.R255W

BCVA, best-corrected visual acuity; F, female, M, male; NA, not available; OD, right eye; OS, left eye; +, wild-type.

Figure 2

Clinical evaluation of patient II:1/family B with typical BVMD. A: Fundus photograph shows a typical vitelliform lesion in both eyes. B, C: Optical coherence tomography (OCT) images demonstrate bilateral subfoveal hyper-reflective material located between the RPE and the neuroretina. D, E: Fundus autofluorescence (FAF) images reveal marked increase in autofluorescence within the vitelliform lesion. OD, right eye; OS, left eye.

In the other four patients from families I–L, typical vitelliform lesions of BVMD at different stages were also observed (Figure 3). For patient II:2 (family K), the EOG showed a decreased Arden ratio (OD 1.20; OS 1.32), while the ERG revealed normal cone and rod responses. The mean age at disease onset of these patients was 12.5 (SD 6.6) years, and BCVA varied widely, ranging from 20/400 to 20/32 (Table 1). Comparison of clinical data between these patients and patients from families A–F revealed no significant differences in age at onset or visual acuity (p>0.05), although age at onset seemed to be younger in these patients. No family history of Best disease was observed in these families.

Figure 3

Fundus photographs of patients with compound heterozygous BEST1 mutations from family I–L. A, B: Fundus photograph of patient II:1/family I reveals a macular scar in both eyes. C, D: Fundus photograph of patient II:2/family J shows a macular scar in the left eye and a vitelliruptive lesion in the right eye. E, F: Fundus photograph of patient II:2/family K demonstrates a bilateral hyperpigmented scar. G, H: Fundus photograph of patient II:1/family L shows a pseudohypopyon lesion in the left eye and a vitelliform lesion in the right eye. OD, right eye; OS, left eye.

Ophthalmologic examinations of patients from families G–H, however, revealed different clinical pictures. The proband of family G (patient II:1) was a 6-year-old boy with a history of macular dystrophy and reduced vision, and his BCVA was 20/200 in the right eye and 20/63 in the left eye at diagnosis. Fundus examination showed subretinal fibrosis in the fovea of the left eye and in the temporal area of both eyes. Multiple yellowish deposits were present in the left eye. OCT demonstrated intraretinal cysts and neurosensory retinal detachment in both eyes. The FAF image showed multiple hyper-autofluorescent lesions in the peripheral retina due to deposits (Figure 4). ERG examination revealed reduced amplitudes of scotopic and photopic full-field ERG responses. The Arden ratio of EOG was 0.95 OD and 1.18 OS. The proband of family H (patient II:1) was referred to Peking Union Medical College Hospital in 2011 at age 23 years with a complaint of decreased vision in both eyes. At examination, BCVA was 20/63 in the right eye and 20/200 in the left eye. Fundus photography revealed a cystoid macular lesion and dozens of round, yellowish, subretinal deposits throughout the posterior pole in both eyes. OCT showed marked intraretinal cystoid fluid collection in the macula and neurosensory retina detachment in both eyes (Figure 5). The amplitudes of the scotopic and photopic full-field ERG responses were decreased. The Arden ratio of EOG was 1.11 OD and 1.23 OS. Based on these findings, the two patients were clinically diagnosed with ARB. Their parents showed no clinical signs of the disease.

Figure 4

Clinical evaluation of patient II:1/family G with homozygous mutation p.M163R. A, B: Fundus examination showed subretinal fibrosis in both eyes with several yellow deposits in the left eye. C, D: Optical coherence tomography (OCT) demonstrated cystoid intraretinal changes and neurosensory retina detachment in both eyes. E, F: The fundus autofluorescence (FAF) image depicts multiple hyper-autofluorescent lesions in the peripheral retina due to deposits. OD, right eye; OS, left eye.

Figure 5

Clinical evaluation of patient II:1/family H with homozygous mutation p.R130L. A, B: Fundus photography revealed a cystoid macular lesion and multiple yellowish subretinal deposits throughout the posterior pole in both eyes. C, D: OCT showed bilateral marked intraretinal cysts in the macula and neurosensory retina detachment. OD, right eye; OS, left eye.

Genetic evaluation

Seven patients from families A–F harbored one heterozygous BEST1 mutation. Based on this finding, along with the typical fundus appearance of BVMD in these patients and the positive family history, an AD inheritance pattern of BVMD was established in these families. Five missense mutations were identified, including two novel mutations (p.T4I and p.A291V) and three reported mutations (p.R218C, p.Q293H, and p.D301G; Table 2). The novel mutations occurred in highly conserved regions (Figure 6).

Table 2

BEST1 mutations identified in the 12 families affected by bestrophinopathy

Exon	Nucleotide change	Amino acid change	Predicted effect	Novel	Hot spot
2	c.11C>T	p.T4I	missense	Yes	No
2	c.38G>A	p.R13H	missense	No	Yes
2	c.97T>C	p.Y33H	missense	Yes	No
4	c.389G>T	p.R130L	missense	Yes	No
5	c.488T>G	p.M163R	missense	Yes	No
5	c.519delA	p.K172Nfs2X	frameshift	Yes	No
5	c.584C>T	p.A195V	missense	No	No
6	c.652C>T	p.R218C	missense	No	No
7	c.763C>T	p.R255W	missense	No	No
7	c.860G>A	p.W287*	nonsense	No	No
8	c.872C>T	p.A291V	missense	Yes	No
8	c.879G>C	p.Q293H	missense	No	Yes
8	c.902A>G	p.D301G	missense	No	Yes

Figure 6

Multiple sequenced alignment of bestrophin-1 around the mutated residues for eight species. The amino acids boxed with colors indicate the positions of novel mutations in the present study. Mutations in a heterozygous state were marked with blue, while mutations in a homozygous or compound heterozygous state were marked with orange. These mutations occurred in highly conserved regions.

Six patients from families G–L carried biallelic mutations in the BEST1 gene. Segregation analysis of the disease in these families along with the negative family history revealed an AR inheritance mode. Compound heterozygous mutations (p.R13H, p.Y33H, p.A195V, p.R255W, p.W287*, and c.519delA) were identified in four patients with BVMD from families I–L, and homozygous mutations (p.M163R and p.R130L) were found in two patients with ARB from families G–H (Table 2). The p.A195V and p.R255W mutations were observed in two patients. To the best of our knowledge, mutations p.Y33H, c.519delA, p.R130L, and p.M163R had not been reported previously, and none were observed in any of the 100 ethnically matched control chromosomes or present in the Single Nucleotide Polymorphism database (SNP) or in the 1000 Genomes Project data set. All the novel mutations occurred in highly conserved regions among different species (Figure 6).

Discussion

In this study, we assessed the phenotypes and genotypes of 13 patients from 12 unrelated Chinese families affected by bestrophinopathy. Mutation analysis revealed biallelic BEST1 mutations in six patients, indicating that patients carrying biallelic BEST1 mutations are common among Chinese bestrophinopathy patients. The detection rate of BEST1 mutations was high, which confirmed the strong association between bestrophinopathy and BEST1 sequence variants. Overall, six novel BEST1 mutations were identified. The high prevalence of novel mutations suggests a difference in the spectrum of BEST1 mutations between Chinese patients and other ethnic groups.

Genes that could cause the same disease in different inheritance modes are not rare. For instance, different mutations in the RHO and RP1 genes can cause adRP or arRP based on their pathogenic effects [13]. In the majority of cases, BVMD is inherited as an AD trait caused by heterozygous mutations in the BEST1 gene, yet AR Best disease has been reported in several families [12-15]. In the present study, families A–F were affected by typical BVMD in an AD inheritance fashion, while families I–L were affected by BVMD in an AR inheritance pattern with segregation of biallelic BEST1 mutations. Clinical data showed no significant differences in onset age or visual acuity between the two groups, although age at onset seemed to be younger in patients with an AR inheritance mode. Our findings are consistent with previous reports and confirm that BEST1 could also cause AR Best disease [12-15].

Patients from family G–H who also carried biallelic BEST1 mutations demonstrated distinct clinical features from those of patients from family I–L. They were diagnosed with ARB based on their unique fundus appearance, reduced rod and cone responses of full-field ERG, and reduced Arden ratios of EOG [16]. Thus, biallelic BEST1 mutations could be associated with at least two phenotypes, BVMD and ARB, which is consistent with other reports of the association of biallelic BEST1 mutations with different clinical pictures, ranging from typical BVMD to ARB [14,23,24].

Mutation analysis revealed five missense mutations in families A–F affected by typical AD Best disease, including two novel mutations (p.T4I and p.A291V) and three reported mutations (p.R218C, p.Q293H, and p.D301G). Exon 8 (amino acid 289–315) harbors 3/5 mutations and has been reported to have a disproportionately higher number of AD Best disease mutations compared with the other coding exons of the gene [25]. Exon 8 encodes for the C-terminal region of bestrophin-1, which interacts with protein phosphatase 2A [26]. The conserved amino acid residues altered by mutations may play a critical functional role in this regulatory interaction.

Six compound heterozygous BEST1 mutations were identified in families I–L with AR Best disease, including p.R13H, p.Y33H, p.A195V, p.R255W, p.W287*, and c.519delA. They are all located outside the four clusters of hot spots defined for AD Best disease (6–30, 80–104, 221–243, and 293–312 amino acid regions) except mutation p.R13H [11], indicating these mutations differ from those that cause typical AD Best disease in mutation locations; thus, a different pathologic mechanism is likely. Interestingly, mutations p.A195V and p.R255W were observed in two patients. Based on our study and a review of the literature [14,27-31], p.A195V is one of the most common mutations identified among patients who carry biallelic mutations. It has been frequently reported in patients with recessive bestrophinopathy in a compound heterozygous state with other mutations, including p.W93P, p.L134V, p.R141H, p.H490del2CTTCA, p.L88del17, and p.Q238L [14,27-31]. The recurrence of p.A195V suggests that certain mutations have a higher predisposition to be pathogenic when present with other mutations on both alleles.

Mutations p.Y33H and c.519delA were novel mutations that had not been previously reported. They occurred in highly conserved regions of bestrophin across multiple species in which alterations may lead to structural or functional changes in the protein. Mutation p.Y33H is predicted to be functionally highly deleterious by the bioinformatic program PolyPhen-2 (score=1.00). Mutation c.519delA is a single base pair deletion that could cause a frame-shift effect and give rise to a premature stop after three nucleotides. This may result in nonsense-mediated decay of the mutant transcript and loss of the protein, leading to the disease.

The other four mutations p.R13H, p.A195V, p.R255W, and p.W287* have already been described in the literature. Mutation p.R13H and p.R255W were reported previously only in patients with BVMD [17,32]. p.W287* was reported in only one patient with atypical Best disease in a compound heterozygous state with L191P, although the description of the phenotype suggested ARB [27]. Mutation p.A195V was reported in patients with BVMD and in patients with ARB carrying another BEST1 mutation on the other allele [14,27-31]. The great clinical variability within our patients along with these data adds to the complexity of phenotypes associated with biallelic BEST1mutations. Due to epistatic effects, any allele can have a different level of gene transcription; thus, in patients who carry biallelic BEST1 mutations, the phenotypic variability could be related to epistatic effects and the interaction of pathogenic effects of different mutations.

Screening for the BEST1 gene revealed two homozygous mutations, p.M163R and p.R130L, respectively, in family G and H affected by ARB. Both were novel mutations in highly conserved regions of bestrophin across multiple species and predicted to be functionally highly deleterious by PolyPhen-2 (score=1.00; 0.99, respectively). Two topology models of human bestrophin-1 were proposed by Tsunenari et al. and Milenkovic et al. [33,34]. The major difference is that Tsunenari et al.’s model shows bestrophin-1 has five transmembrane (TM) domains while the protein is predicted to have four TM domains in the model constructed by Milenkovic et al. However, in both models, homozygous mutations p.R130L and p.M163R associated with the ARB phenotype are located in the non-TM domains of the protein (Figure 7). Twelve of 16 mutations related to ARB are located in the non-TM domains of bestrophin-1 in the two models (Figure 7). This suggests that mutations with non-TM locations are more likely to be associated with the ARB phenotype than mutations residing in TM domains.

Figure 7

Diagrams of human bestrophin-1 summarizing known BEST1 mutations associated with BVMD and ARB phenotypes [10]. A: Protein model of bestrophin-1 proposed by Tsunenari et al. [33]; B: Protein model of bestrophin-1 proposed by Milenkovic et al. [34]. Colored residues indicate a missense mutation or in-frame deletion, while the colored bar indicates a nonsense or frameshift mutation. Mutations in the homozygous or compound heterozygous state reported in the present study are marked with *.

To conclude, we identified six novel and seven previously reported BEST1 mutations in a series of Chinese patients with bestrophinopathy, and described in detail the phenotypes of patients with ARB. To the best of our knowledge, this is the largest study in the literature investigating BEST1 mutations in a Chinese population. This is also the first systematic report of Chinese patients carrying biallelic mutations in the BEST1 gene.