Phenotype and Genotype of Patients with Autosomal Recessive Bestrophinopathy

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1 INTRODUCTION Bestrophin 1 (BEST1) is a member of a family of anion channel proteins. Mutations in the BEST1 gene have been shown to be responsible for Best disease. Best vitelliform macular dystrophy, also known as Best disease is inherited in an autosomal dominant man- ner. The typical clinical features of Best disease are central vitelliform (egg yolk-like) maculopathy and a reduction in the Arden ratio of the electro-oculogram (EOG). The signs of Best disease may be asymmet- ric between the two eyes, there may be multiple fundus abnormalities and the age of onset is highly variable. The genetics of Best disease have been thoroughly investigated. Studies on large pedigrees with Best macular degeneration mapped the disease gene to chromosome 11 [1,2]. More than 180 mutations in the BEST1 gene have been identified in patients from various ethnic populations (HGMD). Mutations in the bestrophin gene are also associated with a spec- trum of ophthalmic conditions including adult onset foveo-macular dystrophy, autosomal dominant vit- reoretinochoroidopathy, autosomal recessive bestro- phinopathy, bull’s eye maculopathy and the MRCS syndrome (microcornea, rod-cone dystrophy, cataract, posterior staphyloma) [3–6]. Bestrophin 1 is a transmembrane protein expressed in the Retinal Pigment Epithelium (RPE). Its expres- sion level is higher in the RPE outside the macula compared to the macular RPE [4]. The function of bestrophin was studied using animal models and transfected cells. These studies suggested that bestrophin functions as a chloride channel activated by calcium and a HCO 3 channel [7–13]. In addition, Bestrophin 1 has also been shown to interact with voltage dependent Ca 2+ channels and modulate cal- cium signaling [14]. Studies on different mutations in bestrophin indicated that the disease pathology could involve a dominant negative effect, haplo-insufficiency Ophthalmic Genetics, 2011, 1–7, Early Online Copyright © 2011 Informa Healthcare USA, Inc. ISSN: 1381-6810 print/ 1744-5094 online DOI: 10.3109/13816810.2011.592172 Received 03 December 2010; revised 11 May 2011; accepted 23 May 2011 Correspondence: Radha Ayyagari, PhD, Associate Professor, Jacobs Retina Center, Room 206, Shiley Eye Center, 9415 Campus Point Drive, La Jolla, CA 92093, USA. Tel: +1 858 534 9029. Fax: +1 858 246 0568. E-mail: [email protected] ORIGINAL ARTICLE Phenotype and Genotype of Patients with Autosomal Recessive Bestrophinopathy Ian M. MacDonald 1 , H. V. Gudiseva 2 , Adda Villanueva 3 , Mark Greve 1 , Rafael Caruso 4 , and Radha Ayyagari 2 1 University of Alberta, Edmonton, Alberta, Canada, 2 University of California San Diego, La Jolla, California, USA, 3 Virtual Eye Care MD, Mexico, and 4 University of Pennsylvania, Philadelphia, Pennsylvania, USA ABSTRACT Purpose: To describe the phenotype and genotype of patients with autosomal recessive bestrophinopathy. Methodology: The phenotype of the subjects was described after a complete ophthalmological examination, and in various cases, ancillary testing of the visual field, optical coherent tomography, full field electroretin- ography and electrophysiology. Genetic analysis was carried out by screening the Bestrophin-1 (BEST1) gene for mutations by dideoxy sequencing and segregation analysis. Results: We identified three previously described mutations (Ala195Val, Leu191Pro and Arg141His) and two potentially pathogenic changes (Trp93Pro and Trp287Ter) in the Best-1 gene. Two patients carried compound heterozygous mutations, Trp93Pro /Ala195Val, and Leu191Pro/Trp287Ter. Two sisters were homozygous for an Arg141His mutation. All individuals with BEST1 gene mutations had signs of maculopathy. Conclusions: Our observations expand the limited number of phenotypes associated with mutations in the Best1 gene. Patients with compound heteroyzygous BEST1 mutations developed atypical forms of Best disease. Two siblings with homozygous Arg141His mutation developed symptoms of typical Best vitelliform dystrophy while their parents had clinical features of mild maculopathy. Keywords: Recessive bestrophinopathy, BEST1 gene, macular dystrophy Ophthalmic Genet Downloaded from informahealthcare.com by University of Alberta on 07/12/12 For personal use only.

Transcript of Phenotype and Genotype of Patients with Autosomal Recessive Bestrophinopathy

1

INTRODUCTION

Bestrophin 1 (Best1) is a member of a family of anion channel proteins. Mutations in the Best1 gene have been shown to be responsible for Best disease. Best vitelliform macular dystrophy, also known as Best disease is inherited in an autosomal dominant man-ner. The typical clinical features of Best disease are central vitelliform (egg yolk-like) maculopathy and a reduction in the Arden ratio of the electro-oculogram (EOG). The signs of Best disease may be asymmet-ric between the two eyes, there may be multiple fundus abnormalities and the age of onset is highly variable.

The genetics of Best disease have been thoroughly investigated. Studies on large pedigrees with Best macular degeneration mapped the disease gene to chromosome 11 [1,2]. More than 180 mutations in the Best1 gene have been identified in patients from various ethnic populations (HGMD). Mutations in

the bestrophin gene are also associated with a spec-trum of ophthalmic conditions including adult onset foveo-macular dystrophy, autosomal dominant vit-reoretinochoroidopathy, autosomal recessive bestro-phinopathy, bull’s eye maculopathy and the MRCS syndrome (microcornea, rod-cone dystrophy, cataract, posterior staphyloma) [3–6].

Bestrophin 1 is a transmembrane protein expressed in the Retinal Pigment Epithelium (RPE). Its expres-sion level is higher in the RPE outside the macula compared to the macular RPE [4]. The function of bestrophin was studied using animal models and transfected cells. These studies suggested that bestrophin functions as a chloride channel activated by calcium and a HCO3 channel [7–13]. In addition, Bestrophin 1 has also been shown to interact with voltage dependent Ca2+ channels and modulate cal-cium signaling [14]. Studies on different mutations in bestrophin indicated that the disease pathology could involve a dominant negative effect, haplo-insufficiency

Ophthalmic Genetics, 2011, 1–7, Early OnlineCopyright © 2011 Informa Healthcare USA, Inc.ISSN: 1381-6810 print/ 1744-5094 onlineDOI: 10.3109/13816810.2011.592172

Received 03 December 2010; revised 11 May 2011; accepted 23 May 2011Correspondence: Radha Ayyagari, PhD, Associate Professor, Jacobs Retina Center, Room 206, Shiley Eye Center, 9415 Campus Point Drive, La Jolla, CA 92093, USA. Tel: +1 858 534 9029. Fax: +1 858 246 0568. E-mail: [email protected]

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ORIGINAL ARTICLE

Phenotype and Genotype of Patients with Autosomal Recessive Bestrophinopathy

Ian M. MacDonald1, H. V. Gudiseva2, Adda Villanueva3, Mark Greve1, Rafael Caruso4, and Radha Ayyagari2

1University of Alberta, edmonton, Alberta, Canada, 2University of California san Diego, La Jolla, California, UsA, 3Virtual eye Care MD, Mexico, and 4University of Pennsylvania, Philadelphia, Pennsylvania, UsA

ABSTRACT

Purpose: To describe the phenotype and genotype of patients with autosomal recessive bestrophinopathy.

Methodology: The phenotype of the subjects was described after a complete ophthalmological examination, and in various cases, ancillary testing of the visual field, optical coherent tomography, full field electroretin-ography and electrophysiology. Genetic analysis was carried out by screening the Bestrophin-1 (Best1) gene for mutations by dideoxy sequencing and segregation analysis.

Results: We identified three previously described mutations (Ala195Val, Leu191Pro and Arg141His) and two potentially pathogenic changes (Trp93Pro and Trp287Ter) in the Best-1 gene. Two patients carried compound heterozygous mutations, Trp93Pro /Ala195Val, and Leu191Pro/Trp287Ter. Two sisters were homozygous for an Arg141His mutation. All individuals with Best1 gene mutations had signs of maculopathy.

Conclusions: Our observations expand the limited number of phenotypes associated with mutations in the Best1 gene. Patients with compound heteroyzygous Best1 mutations developed atypical forms of Best disease. Two siblings with homozygous Arg141His mutation developed symptoms of typical Best vitelliform dystrophy while their parents had clinical features of mild maculopathy.

Keywords: Recessive bestrophinopathy, Best1 gene, macular dystrophy

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or total loss of function mechanisms depending on the nature of protein change [6,10]. Mutations that cause early-onset disease have been considered to be the result of a nonfunctional protein and a dominant nega-tive effect. Mutations associated with adult onset Best disease were reported to have reduced chloride channel function [12]. In addition, the variation observed in phe-notype was also attributed to the effect of the mutation on the chloride channel function of bestrophin 1 versus the effect on its calcium channel activity [15].

Significant variability was observed in the penetrance and expressivity of bestrophin 1 mutations. Severity of disease, age of onset and the symptoms were found to vary within and between families with a single geno-type [16]. Some of the patients with late-onset Best dis-ease may have a misdiagnosis of age-related macular degeneration (AMD) because of the overlap in clinical symptoms and age of onset [4,6].

Recessive bestrophinopathy due to either compound heterozygous or homozygous Best1 gene mutations has been reported [17]. Several mutations associated with recessive bestrophinopathy were also reported to be involved in causing dominant Best disease when they were present in the heterozygous state [3,6,18–20]. The clinical phenotype of some patients with recessive bestrophinopathy is distinct from that seen in Best dis-ease while in others it is similar to the typical phenotype observed in autosomal dominant vitelliform dystrophy [21,22]. Identification of additional families with reces-sive bestrophinopathy and detailed characterization of the clinical phenotype of homozygous and heterozy-gous individuals will assist in establishing phenotype-genotype correlations.

In this manuscript, we describe the genotype and phenotype of patients with recessive bestrophinopathy due to either compound heterozygous or homozygous bestrophin 1 mutations. In these patients we identi-fied three previously described mutations and two potentially pathogenic changes in the Best1 gene. The clinical phenotype of Best disease in parents of affected siblings is milder compared to patients with recessive mutations. These observations expand the spectrum of phenotypes associated with bestrophin 1 mutations and may assist in establishing phenotype-genotype cor-relations and understand the molecular pathology of bestrophinopathies.

MATERIALS AND METHODS

Clinical Studies

Subjects were from three different unrelated families. All research protocols were in accordance with Institutional guidelines and the Declaration of Helsinki.

The clinical phenotype was characterized by measur-ing visual acuity, testing visual fields, examining the fundus, estimating dark-adapted visual thresholds, and

recording the electroretinogram (ERG), and electro-ocu-logram (EOG)[23] according to ISCEV standards (www.iscev.org). The normal range of the Arden ratio of the EOG (ratio of the light peak/dark trough) is more than 1.65 for our laboratory.

Genetic Analysis

Mutation analysis of the Best1 gene was carried out by amplification of all 11 exons by PCR using prim-ers reported previously [1,16]. Mutation analysis was carried out by sequencing these PCR products by dideoxy sequencing using an ABI 3100 sequence analyzer as described earlier [24]. The presence of all novel non-synonymous sequence changes were ana-lyzed in a minimum of 98 chromosomes from unre-lated unaffected individuals. The impact of these novel non-synonymous changes on Bestrophin protein was determined using POLYPHEN and SIFT programs (http://genetics.bwh.harvard.edu/pph2/, http://sift.jcvi.org/).

RESULTS

Phenotype of Patients Studied

Case IA 28-year-old Caucasian female with atypical Best dis-ease with a bifocal lesion was studied. A macular cystoid lesion with discrete yellowish deposits in its periphery, connected with a temporal paramacular area with less elevation, was observed by clinical examination (Fig. 1). Optical coherent tomography (OCT) revealed a large central cystoid space and delamination of the retinal layers.

Case IIA 44-year-old female of Asian (Fillipino) origin with typical Best disease was diagnosed at age 36. Fundus imaging showed a vitelliform macular lesion with fibrosis and multiple discrete vitelliform deposits in the perifovea (Fig. 2). OCT revealed a minimal cleft at the RPE- photoreceptor interface (no cystoid space).

Cases III and IVA family was identified with two female children affected by early onset Best macular dystrophy at the ages of 11 and 6 years (Fig. 3). Both affected sisters at ages 14 and 8 had a normal full field ERG (data not shown). The fundus of the older affected child had circu-lar RPE disruption with fibrosis and yellowish deposits in the macula (Fig. 4). Despite the obvious maculopathy, color vision as measured with the D15 test was normal in both eyes and best corrected vision was recorded as 20/200 (OD) and 20/20 (OS). The waveforms of her full

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field ERG as recorded with DTL electrodes according to the ISCEV standards (www.iscev.org) were normal. The Arden ratio of the electro-oculogram recorded at the same age was 1.25 (OD) and 1.44 (OS); normal for our laboratory is 1.65 to 1.6. Optical coherent tomographic

imaging was not available. The younger sister had a best corrected visual acuity of 20/20 (OU). Based on the clinical presentation and the EOG, a clinical diag-nosis of Best vitelliform macular dystrophy was made. The absence of typical features of Best disease in either

FIGURE 1 Fundus photograph of the left and right eye of the 28-year-old female (Case I) showing a cystoid macular lesion.

FIGURE 2 Fundus photograph of the left and right eye of the 44-year-old female (Case II) showing a vitelliform macular lesion and multiple smaller vitelliform lesions beyond the central macula.

FIGURE 3 Pedigree of Cases III and IV. Squares are males. Circles are females. Filled symbols indicate bestrophinopathy. Open symbols indicate absence of bestrophinopathy. Half filled symbols indicate mild maculopathy.

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parent was unexpected. The father at age 40 had a mild maculopathy; the Arden ratios of the EOG were recorded as 1.96 (OD) and 2.15 (OS) (Fig. 5). The mother at age 37 had a pigmentary maculopathy in the left eye; the Arden ratio of the EOG was 1.95 (OD), 1.99 (OS) (Fig. 5). This family had three more children who were clinically normal when examined at ages 14, 12, and 5.

Genotype of Patients Studied

Mutation analysis of the two unrelated patients and the two sisters revealed the presence of either previously reported mutations or potentially pathogenic sequence alterations in the Best1 gene. All the pathogenic or potentially pathogenic changes observed in these patients occurred either in a compound heterozygous or homozygous state.

Case IIn this patient with atypical Best disease, a Leu191Pro missense change and a Trp287Ter nonsense change were detected in the Best1 gene. Both the sequence changes detected have not been reported in the Caucasian

population. We have previously listed these two changes in a publication describing molecular diagnostic testing [25]. The Trp287Ter mutation results in premature trun-cation of the protein from 585 amino acids to 287 amino acids. This will result in a complete loss of the cytosolic loop of bestrophin 1 [26]. It is also possible that this change may result in nonsense-mediated decay of the mutant transcript and loss of the protein.

Case IITwo missense mutations, Trp93Pro and Ala195Val mutations were found in the Best1 gene in a patient with later onset Best disease (diagnosed at age 36). The Trp93Pro change is a novel change and the Ala195Val missense change has been previously reported as a mutation.

Cases III and IVSequencing the Best1 gene in the two affected sisters and their parents identified a homozygous change Arg141His in both sisters. Both parents of these affected siblings and another normal sibling carry the Arg141His change in the heterozygous state. This change has been reported as a pathogenic mutation in other patients. To the best of our knowledge this is the first report of this mutation segregating in the homozygous state. This mutation is present in the heterozygous state in another unaffected sibling and two more siblings do not have this sequence change.

DISCUSSION

In this study we describe the phenotype and genotype of individuals carrying heterozygous, homozygous or compound heterozygous mutations in the Best1 gene. Four patients with recessive retinal dystrophy carried three previously reported mutations and two additional changes identified as damaging changes by PolyPhen and SIFT programs. One patient had Trp93Pro and Ala195Val as compound heterozygous mutations, another patient had Leu191Pro and Trp 287Ter changes and two sisters carried the Arg141His mutation in the homozygous state. The older family members carrying

FIGURE 4 Fundus photograph of the right eye of the other affected child showing apparent central disciform lesion with pigment, subretinal scarring and lipid deposition.

FIGURE 5 Fundus photograph of left eye of the mother and left eye of the father showing minimal macular changes at age 37 and 40, respectively.

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the Arg141His change in the heterozygous state devel-oped mild clinical signs of maculopathy. These obser-vations confirm the involvement of the Best1 gene in recessive macular dystrophy in the set of patients we studied.

In the family with two daughters carrying homozy-gous mutations in the Best1 gene, the phenotype of the daughters is relatively early in terms of its onset, but has not progressed to that of previously reported patients with severe functional loss described in auto-somal recessive bestrophinopathy.16 The parents would not have come to our attention had their daughters not been diagnosed with a maculopathy and would per-haps not have been identified with a maculopathy until later in life. This example illustrates the importance of genotype-phenotype correlations; one cannot presume that homozygous mutations in the Best1 gene will result in a severe phenotype.

The Trp93Pro and Ala195Val mutations were observed in one affected individual (Case II) with no family his-tory of Best disease. The Trp93Pro is a novel change which was not observed in control samples analyzed in our laboratory or in other laboratories. Earlier we reported a missense mutation altering the tryptophan at position 93 to cysteine in a dominant pedigree with four affected members [16]. In addition, this mutation was also reported in two other patients with dominant Best macular degeneration [1,27] The tryptophan at position 93 is conserved across multiple species [1].

Detailed studies were carried out on the Trp93Cys Bestrophin mutation using cultured cells, patient tis-sue and rat RPE cells [14,28,29]. Furthermore, knock-in mouse models carrying the W93C Bestrophin muta-tion have also been described [14]. Although Best1 is thought to function as a Ca2+-activated Cl− channel, the RPE cells from Best1+/W93C mice exhibited normal Cl− conductance. The ATP-stimulated changes in [Ca2+]i in RPE cells from Best1+/W93C and Best1W93C/W93C mice were suppressed compared to the cells from littermate wild type controls. These studies indicated the presence of bestrophin in the RPE of a patient with the homozygous Trp93Cys change and correct trafficking of the mutant Bestrophin to the basolateral plasma membrane of the RPE suggesting that the Trp93Cys mutation alone does not result in a null phenotype or alter the intracellular trafficking of the protein. This mutation was reported to exert a dominant negative effect on its chloride channel function [13,30,31]. Although the Trp93Pro and Trp93Cys changes involve the same amino acid, the effect of having proline vs. cysteine at that position is not known. Tryptophan at position 93 is highly con-served suggesting that it is critical for the normal func-tion of Bestrophin. Our patient carrying the Trp93Pro is of Filipino origin and the chances of this change being a rare polymorphism in that population cannot be ruled out.

A second mutation, Ala195Val, was found in the Filipino subject (Case II) with the Trp93Pro change. The

Ala195Val mutation was reported previously in patients with dominant vitelliform dystrophy [6,19]. Two differ-ent topology models have been proposed for human bestrophin. Based on these models, the Ala195Val muta-tion is located either in the 4th transmembrane domain or the cytoplasmic loop of bestrophin. The effect of this mutation on the anion channel activity of bestrophin has not been tested. A similar Alanine to Valine change at codon 243 (A243V) observed in patients with adult vitelliform dystrophy alters chloride channel activity. In vitro functional studies indicated that this mutation does not exert a dominant negative effect [12]. As observed in case of the A243V mutation, the Ala195Val mutation may also result in a null allele and this mutation along with the Trp93Pro may cause recessive Bestrophinopathy. Functional studies are needed to evaluate the affect of Ala195Val and Trp93Pro changes and the mechanism underlying the disease pathology in this patient. Parents of this patient were not available to study the effect of the Trp93Pro mutation in the heterozygous state.

The Leu191Pro change observed in another subject (Case I) is located in the 4th transmembrane domain based on the topology model proposed by Tsunenari [32]. The Leucine at position 191 is a conserved amino acid and it is possible that this change alone is pathogenic or may be in combination with the second Trp287Ter change observed in this patient. This second mutation in this patient is a nonsense mutation in exon 7 which is predicted to truncate the protein resulting in loss of the cytoplasmic loop. This change was not detected in 100 control samples. It is likely that the nonsense change alone could be causative. Two other truncating muta-tions in Bestrophin have been previously reported in patients with dominant vitelliform dystrophy [5,19]. It is likely that this mutation may lead to nonsense medi-ated decay of the mutant transcript. The parents of this patient were not available for evaluation.

In one family we observed the missense mutation Arg141His in the homozygous state in two affected sisters. Both parents carrying this change in the heterozygous state had mild maculopathy. This change in the heterozygous state was reported as a muta-tion in patients with dominant vitelliform dystrophy [3,19]. This mutation was also found as a compound heterozygous change in combination with Val317Met or Pro152Ala or Leu41Pro in patients with recessive retin-opathy [17]. The Cl- channel activity of cells expressing mutant bestrophin with the Arg141His mutation was depleted and found to be much lower compared to the Cl- channel activity of cells co-expressing the wild type and mutant bestrophin [17]. This suggests that the Arg141His mutation in the homozygous state may result in a null phenotype. Onset of vision loss in these patients was reported to vary between ages 4 and 40 years [17]. The sisters with the homozygous mutation were diagnosed with typical Best macular dystrophy in the first decade of life. Their parents, heterozy-gous carriers of the same mutation, had mild retinal

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changes in their late 30s and early 40s suggesting that this change in the homozygous state may affect the retina more significantly compared to its presence in the heterozygous state. To our knowledge, this is the first report of homozygous Arg141His mutations in the Best1 gene causing typical Best vitelliform dystrophy, with mild clinical features in the heterozygous carri-ers. A patient, age 4, with compound heterozygous mutations Arg141His/Leu41Pro was reported to have a more severe phenotype with macular scars, diffuse RPE changes, an abnormal full field and pattern ERG and EOG (absent light rise). That patient’s phenotype contrasts sharply with Case III (age 10) who had a central maculopathy only, with a normal ERG and abnormal EOG, in keeping with clinically typical Best disease. Bakall et al. reported that the clinical symptoms in a patient carrying the Trp93 Cys in the homozygous state were not more severe than his relatives carrying the same mutation in the heterozygous state [29]. It is also interesting to note that the onset of disease in heterozygous carriers with the Trp93Cys mutation is earlier than the patient with the same mutation in the homozygous state.

None of the patients described in the literature with two mutations in the Bestrophin gene had vitelliform lesions [33]. Similarly, case III and IV did not show a vitelliform change, but had a later stage of Best dis-ease, stage 4c (fibrous scarring with apparent choroidal neovascularization and hemorrhage) at a young age [34]. Their fundus angiography showed widespread patchy hyperfluorescence. The EOG showed a severely reduced light rise (reduced Arden ratio) and a signifi-cant reduction in the amplitudes of the pattern ERG. A relative of a recessive retinal dystrophy patient was reported to be clinically normal at age 55, despite car-rying the Arg141His mutation [4,17]. This mutation involves a conserved amino acid [19]. Among the cases presented in this study, the patient with the Trp93Pro and Ala195Val compound heterozygous changes and the sisters homozygous for the Arg141His mutation had typical symptoms of Best vitelliform dystrophy whereas Case I with Leu191Pro and Trp287Ter had atypical Best macular degeneration.

Bestrophin is an anion channel and most of the muta-tions found in this protein are dominant mutations [31]. Bestrophin oligomerizes to form dimers or tetramers [13,35]. Heterozygous mutations may result in the for-mation of defective channels composed of wild type and mutant subunits [13]. Haploinsufficiency has been suggested to be the possible mechanism underlying Best disease for patients with the Ala243Val and Ala260Phe mutations [31].

Variation in the expression of clinical symptoms was observed among patients within a family or patients from different families carrying the same Bestrophin mutation [16]. This could be due to the variable pene-trance or the presence of factors that modify the severity of Best disease. Detailed studies on the phenotype and

genotype of additional patients with homozygous and heterozygous Bestrophin mutations will aid in under-standing the effect of these mutations and presence of potential modifying factors.

ACKNOWLEDGMENTS

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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