- Email: [email protected]
Assessment of mutations in the best macular dystrophy (VMD2) gene in patients with adult-onset foveomacular vitelliform dystrophy, age-related maculopathy, and bull’s-eye maculopathy1
Assessment of Mutations in the Best Macular Dystrophy (VMD2) Gene in Patients with Adult-onset Foveomacular Vitelliform Dystrophy, Age-related Maculopathy, and Bull’s-eye Maculopathy Johanna M. Seddon, MD, ScM1,2 Mehran A. Afshari, MD, MPH,1 Sanjay Sharma, MD, MSc (Epid),1,2 Paul S. Bernstein, MD, PhD,3 Sandy Chong, BS,2 Amy Hutchinson, BA,4 Konstantin Petrukhin, PhD,5 Rando Allikmets, PhD4,6 Purpose: To study the presence of Best macular dystrophy (VMD2) gene mutations in patients diagnosed with maculopathies other than classic Best disease and to describe the clinical characteristics of these subjects. Design: Case-comparison study of phenotype-genotype correlations. Methods: Patients with either age-related maculopathy (ARM; n ⫽ 259) or maculopathies other than classic Best disease (n ⫽ 28) were screened for mutations in the Best gene (VMD2; OMIM 153700). These cases were compared with ethnically similar subjects in the same age range without maculopathy (n ⫽ 196). All patients underwent a complete dilated ocular examination, and all affected individuals underwent fundus photography. Phenotype-genotype comparisons were made. Main Outcome Measures: Presence of mutations in the Best gene (VMD2; OMIM 153700) and the clinical phenotype. Results: Three of 259 patients (1%) with ARM and 2 of 28 patients (7%) with other maculopathies including 1 of 3 patients with adult-onset foveomacular vitelliform dystrophy and 1 of 5 patients with a bull’s eye maculopathy, but none of the controls, were found to possess amino acid-changing variants in the VMD2 gene. These included a man with confluent drusen and retinal pigment epithelial detachments (variant in exon 6; T216I), a man with geographic atrophy and numerous soft drusen (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations resembling bull’s-eye maculopathy (variant in exon 4; E119Q), and a woman diagnosed with adult-onset foveomacular vitelliform dystrophy (variant in exon 4; A146K). Conclusions: Novel mutations in the VMD2 gene were found in patients diagnosed with maculopathies other than classic Best disease. Some cases diagnosed as adult-onset vitelliform foveomacular dystrophy may represent a variant of Best disease with delayed onset. The VMD2 gene does not play a major role in the development of ARM. Ophthalmology 2001;108:2060 –2067 © 2001 by the American Academy of Ophthalmology.
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Originally received: June 14, 1999. Accepted: June 4, 2001. Manuscript no. 99307. 1 Department of Ophthalmology, Massachusetts Eye Ear Infirmary, Harvard Medical School, Boston, Massachusetts. 2 Epidemiology Unit, Massachusetts Eye Ear Infirmary, Harvard Medical School, Boston, Massachusetts. 3 Department of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah. 4 Department of Ophthalmology, Columbia University, New York, New York. 5 Department of Human Genetics, Merck Research Laboratories, West Point, Pennsylvania.
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Department of Pathology, Columbia University, New York, New York. Presented as a poster at the American Academy of Ophthalmology annual meeting, Orlando, Florida, October 1999. Supported in part by the National Eye Institute, Bethesda, Maryland (grant nos.: EY11309 [JMS] and EY11600 [PSB]; Research to Prevent Blindness, Inc., New York, New York; an RPB Lew R. Wasserman Merit Award (JMS); an RPB career development award (PSB and RA); the Lions Eye Research Fund, Inc., Northboro, Mass., an unrestricted grant to Moran Eye Center; and The Ruth and Milton Steinbach Fund, New York, NY. The authors have no proprietary interest in the products or devices mentioned herein. Correspondence and reprint requests to Johanna M. Seddon, MD, Department of Ophthalmology, Epidemiology Unit, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114. E-mail: [email protected]. ISSN 0161-6420/01/$–see front matter PII S0161-6420(01)00777-1
Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease Best disease is an autosomal dominant macular dystrophy characterized by the early onset of vitelliform macular lesions. Patients may be asymptomatic or have visual loss at the time of presentation.1 The fundus findings of patients with Best disease can be classified into five stages: no demonstrable pathologic features with abnormal Arden ratios on electro-oculography (EOG) (stage 0), mild retinal pigment epithelial disturbance presenting as window defects on fluorescein angiography (stage 1), vitelliform lesion and scrambled egg appearance (stage 2), pseudohypopyon (stage 3), orange-red atrophic lesion (stage 4a), macular scarring (stage 4b), and neovascularization (stage 4c).2 In Best disease, the results of EOG are abnormal, whereas the electroretinogram results are typically normal.3 Adult-onset foveomacular vitelliform dystrophy is a disorder characterized by the presence of a small, round, yellow subretinal lesion, with a central pigmented spot, in a middle-aged patient.4,5 Drusen may be present with this lesion. On angiography, the central pigmented areas are associated with relative hypofluorescence, whereas the surrounding “vitelliform” material is associated with hyperfluorescence. The EOG Arden ratios in patients with adultonset foveomacular dystrophy are usually normal or minimally subnormal. This disease is typically associated with minimal visual loss. Histologically, these lesions correspond to large amounts of lipofuscin within retinal pigment epithelial (RPE) cells, pigment-laden macrophages in the retina, focal loss of photoreceptors, and RPE cell atrophy.6 There are some phenotypical similarities among Best disease, adult vitelliform maculopathy, and the more common problem of age-related maculopathy (ARM). There is also increasing interest in the hypothesis that ARM may be, in part, a genetic disease. Scientific evidence that ARM has a genetic basis comes from numerous studies. A recent case-control study quantified that macular degeneration is 2.5 times more common among first-degree relatives of case probands, when compared with first-degree relatives of control probands.7 This familial aggregation was confirmed in a population-based study by Klaver et al.8 A few small twin studies have estimated a higher concordance rate between monozygotic twins than dizygotic twins.9,10 Segregation studies suggest that there is a single major gene defect in a large proportion of affected individuals.11 The TIMP3 gene, responsible for Sorsby’s fundus dystrophy, was ruled out as a candidate gene for ARM.12 However, Allikmets et al13 reported that ARM patients, particularly those with dry ARM, had a higher prevalence of mutations in the Stargardt gene (ABCR) when compared with controls. This observation has been investigated in other study populations.14,15 In a recent report analyzing a large family of patients with macular degeneration, the disease locus for this particular family mapped to chromosome 1q25-q31 between markers D1S466 and D1S413.16 Age-related maculopathy has a myriad of manifestations that may be pathogenetically distinct. Clinical signs of ARM include: drusen, RPE hyperplasia or depigmentation (collectively known as early ARM), and geographic atrophy, RPE detachment, and choroidal neovascularization (collec-
tively known as advanced ARM or age-related macular degeneration [AMD]).17 Bull’s-eye maculopathy refers to the ophthalmoscopic finding of depigmentation of central retinal pigment epithelium surrounded by a relatively normal area, giving a bull’seye appearance to the macula. This phenotype is associated with a number of macular diseases, including chloroquine and hydroxychloroquine maculopathies, cone dystrophy as well as Stargardt disease, benign concentric annular dystrophy, and a variety of other macular dystrophies.18 –20 The gene causing Best macular dystrophy (VMD2) has recently been cloned and is located on chromosome 11 at 11q13.21 Petrukhin et al21 identified five independent disease-specific mutations in five families with Best disease. These mutations were associated with nucleic acid changes at exons 2, 4, 6, and 8. Marquardt et al22 also reported mutations in this gene (VMD2) in patients with Best disease. The Best gene is heavily expressed in the retina, specifically in the RPE. Because the gene associated with Best disease is a good candidate for other maculopathies, we screened the DNA of patients and controls in our databases for the presence of variants in this gene. We recently reported that novel mutations in the VMD2 gene were found in some of our patients with Best macular dystrophy as well as other maculopathies not diagnosed with classic Best disease.23 In the present study, we describe in detail the clinical features and phenotypes of the patients diagnosed with maculopathies other than Best disease, as well as some of their family members, and list their variant sequences in the VMD2 gene. Results suggest that different mutations in a single gene can result in a variety of phenotypes and support the likelihood that emerging new genetic information will modify definitions for the clinical diagnosis of various maculopathies.
Materials and Methods The Family Macular Degeneration Study is a National Institutes of Health-supported, Massachusetts Eye and Ear Infirmary Human Subject Committee-approved study being performed to determine the genetic basis of macular degeneration. As part of this study, patients are asked questions about their family members, and a complete pedigree is constructed. All participants consent to a thorough ocular evaluation with fundus photography, which may also include diagnostic testing when appropriate (fluorescein angiography, electrophysiologic testing, or both). They also provide blood and buccal swab sources of DNA to be used for genetic testing. From the Massachusetts Eye and Ear Infirmary site, we screened the DNA samples from 188 patients with ARM who were classified in a standardized manner as previously described [Seddon et al. Invest Ophthalmol Vis Sci 38(Suppl):S675, 1997]: 155 (82%) with the “dry” type of the disease, including extensive intermediate drusen, large drusen, or geographic atrophy, and the remaining 33 (18%) with the “wet,” or exudative form with fluorescein angiographic evidence of choroidal neovascularization, and 28 patients with maculopathies other than classic Best disease. These 28 cases included myopic retinal degeneration (n ⫽ 1), macular RPE changes or RPE detachment of uncertain cause in young patients (n ⫽ 2), cone dystrophy (n ⫽ 4), multifocal RPE detachment consistent with a diagnosis of multifocal Best disease
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Figure 1. Patient 1. A, Drusenoid-type of retinal pigment epithelial detachment, right eye. B, Geographic atrophy corresponding to previous area of retinal pigment epithelial detachment, right eye.
Figure 2. Patient 2. Numerous soft drusen with central area of geographic atrophy, right eye.
Figure 3. Patient 3. Soft drusen and central area of retinal pigment epithelial alteration associated with extramacular drusen, left eye.
Figure 4. Patient 4. Few drusen associated with a ring like area of retinal pigment epithelial depigmentation, resembling a bull’s eye, left eye.
Figure 5. Patient 5. A, Symmetric, slightly elevated yellow lesion with central hyperpigmentation consistent with adult-onset foveomacular vitelliform dystrophy. Fine drusen are also present, right eye. B, Fluorescein angiogram demonstrating the ring of hyperfluorescence with hypofluorescence centrally and late staining of the central vitelliformlike lesion, right eye.
Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease (n ⫽ 1), atypical retinitis pigmentosa (n ⫽ 1), Stargardt disease (n ⫽ 2), bull’s-eye maculopathy (n ⫽ 5), pattern dystrophy (n ⫽ 5), early onset of drusen (n ⫽ 1), RPE changes with family history of pattern dystrophy (n ⫽ 1), annular pigment dystrophy (n ⫽ 2), and adult-onset foveomacular vitelliform dystrophy (n ⫽ 3). DNA samples from an additional 71 patients with ARM from the Moran Eye Center in Utah were also screened. All of these subjects signed consent forms to participate in the research protocol. They were classified in the same manner as the ARM cases at the Massachusetts Eye and Ear Infirmary, and 10 cases (14%) had the exudative form of the disease. The control group consisted of 196 white subjects of similar ethnicity and age range as the cases (ages 65 years and older)who were examined by two of the authors (JMS and PSB) and found not to have ARM or any other maculopathy. All 11 exons of the VMD2 gene, as well as 5⬘ and 3⬘ untranslated regions, were screened for variants using single-strand conformation polymorphism analysis and direct sequencing of variants in all patients and controls as described previously.21,24 Five patients were identified with amino acid-changing variants in the VMD2 gene, and none of these variants were found in the 196 controls (392 chromosomes). All other variants found in the VMD2 gene, including several common alleles, have been described elsewhere.23 The purpose of this report was to describe the clinical features of these five cases and some of their family members.
Results Case Reports Patient 1. A 72-year-old male noted mild gradual visual deterioration over a 13-year period. The patient reported no family history of eye problems or visual loss. In February 1995, his visual acuity was recorded as 20/25 in each eye. His anterior segment examination results were within normal limits. Funduscopic examination revealed large confluent drusen and hypopigmentation and clumping of RPE in both eyes. There was no evidence of basal laminar drusen in either eye. Fluorescein angiography demonstrated staining of drusen, but no evidence of choroidal neovascularization. The patient was seen again in July 1996. At that time, his visual acuity was 20/32 in the right eye and 20/40 in the left eye. Examination of the fundus revealed bilateral confluent drusen and RPE detachments (Fig 1A, right eye).5 Fluorescein angiography demonstrated staining of drusen and pooling of dye in the area of the RPE detachments. There was no evidence of choroidal neovascularization. When the patient was examined again in June 1998, visual acuity was 20/25 in the right eye and 20/50 in the left eye. On ophthalmoscopic examination, geographic atrophy in the right eye and large drusemoid-type RPE detachment in the left eye were present (Fig 1B, right eye). Electro-oculography revealed normal Arden ratios of 3.2 in the right eye and 3.4 in the left eye. A 751C 3 T variant was found in exon 6 of the VMD2 gene, resulting in a T216I mutation. Patient 2. A 76-year-old male described gradual visual loss over a 10-year period. The patient reported no family history of eye problems or visual loss. In September 1993, the patient’s visual acuity was 20/40 in the right eye and finger counting in the left eye. He was pseudophakic in the right eye and had a moderate cataract in the left eye. He was noted to have numerous soft confluent drusen in the right eye and numerous soft drusen with geographic atrophy in the left eye. There were no extramacular drusen or peripheral reticular pigmentary changes. The patient underwent uncomplicated cataract extraction with intraocular lens implantation in the left eye a few months after initial assessment. His visual acuity during his most recent examination (September 1998) was 20/40 in the right eye and 20/250 in the left eye. His
funduscopic examination revealed numerous soft drusen with some degree of confluence and geographic atrophy in both eyes (Fig 2; right eye). Fluorescein angiography revealed hyperfluorescence of the drusen and late frame hyperfluorescence and staining associated with the area of geographic atrophy. Electro-oculography revealed Arden ratios of 3.3 in the right eye and 2.5 in the left eye. A 1803C 3 T (L567F) variant was found in exon 10 of the VMD2 gene. Patient 3. A 61-year-old female noted a mild gradual visual deterioration in the right eye over a 4-year period. The patient reported no family history of eye problems or visual loss. Her visual acuity was 20/25 in each eye. Fundus examination demonstrated small and intermediate drusen in the right eye and large soft drusen in the left eye, consistent with dry ARM, as well as extramacular drusen (Fig 3, left eye). No reticular pigmentary changes were noted in the periphery of either eye. Fluorescein angiography revealed hyperfluorescence of the drusen and window defects consistent with the areas of RPE alteration. Three years later, visual acuity was 20/25 in the right eye and 20/32 in the left eye, and funduscopic examination and fluorescein angiogram were unchanged. The same variant as patient 2, 1803C 3 T (L567F) was found in exon 10 of the VMD2 gene in this patient. The EOG results were normal and revealed Arden ratios of 2.0 in the right eye and 2.1 in the left eye. Two half-siblings (same father) also underwent genotyping: a male age 46 and a female age 50. The female was examined and had no evidence of maculopathy and the male reported no eye disease. Neither relative had the mutation in the VMD2 gene. Patient 4. A 77-year-old female noted a gradual decline in her vision over a 4-year period. The patient reported no family history of eye problems or visual loss. She had a history of systemic lupus erythematosus and had been taking medication of uncertain type in the past 10 years. A thorough search of her past records revealed no history of chloroquine or hydroxychloroquine therapy. Her visual acuity was 20/50 in the right eye and 20/640 in the left eye. Funduscopic examination revealed early annular RPE depigmentation in the right eye and an annular area of retinal pigment depigmentation, consistent with a bull’s-eye maculopathy in the left eye (Fig 4). Reticular pigmentary changes were noted in both eyes, without signs of extramacular drusen. Angiography revealed a central area of hypofluorescence surrounded by an annular ring of hyperfluorescence. There was no evidence of either a “dark choroid” or other lesions on angiography. Electro-oculography could not be performed because the patient is deceased. A 459G 3 C (E119Q) mutation was detected in exon 4 of the VMD2 gene. Patient 5. A 60-year-old female noted mild visual deterioration in the right eye over a 6-year period. Her visual acuity was 20/32 in the right eye and 20/20 in the left eye. Her ophthalmoscopic examination revealed a small RPE detachment with central hyperpigmentation in the right eye consistent with the diagnosis of adult-onset foveomacular vitelliform dystrophy (Fig 5A). Mottling of the RPE and fine drusen were also seen. The photographs and fluorescein angiograms were reviewed independently by two other retina specialists who agreed with the clinical diagnosis of adultonset vitelliform macular dystrophy. Mild reticular pigmentary changes were noted in the periphery of the right eye, without extramacular drusen. Fluorescein angiography in the right eye revealed relative hypofluorescence corresponding to the central area of hyperpigmentation and late frame staining in the area of deposition of vitelliform material (Fig 5B). Electro-oculography revealed abnormal Arden ratios of 1.01 in the right eye and 1.5 in the left eye. Sequencing revealed a 540GC 3 AA (A146K) mutation in exon 4 of the VMD2 gene in this patient. This patient informed us that her deceased brother (Fig 6) had macular degeneration, which was diagnosed at age 67. After obtaining consent from the family, we obtained his ocular photo-
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Ophthalmology Volume 108, Number 11, November 2001 graphs and fluorescein angiogram, which demonstrated that he had maculopathy in both eyes consisting of a ring of yellow deposits in the right eye and central pigment alteration in the left eye. Angiographically, the lesion in his left eye had a nonfluorescent center surrounded by a ring of hyperfluorescence, which are features resembling adult-onset foveomacular vitelliform dystrophy. The mother in this family had decreased central vision and a history of maculopathy. The three sons of the proband (ages 46, 52, and 53) underwent genotyping, and two were found to have the same variant in the VMD2 gene and one (age 52) did not. This latter son was examined and photographed and did not have any evidence of maculopathy. Thus far, we have been unable to arrange an ocular examination, photography, or EOG for the two sons with the variants in the Best gene.
Discussion Four unique variants were detected in the VMD2 (Best) gene in 5 patients with maculopathies: 3 of 259 subjects (approximately 1%) with age-related maculopathy, and 2 of 28 other subjects (approximately 7%) with maculopathies not diagnosed as classic Best disease. These two latter cases with genetic variants in VMD2 included one of three subjects with adult-onset foveomacular vitelliform maculopathy and one of five subjects with bull’s-eye maculopathy. It is interesting to note that three of these five patients (patient 1, confluent drusen type of RPE detachment; patient 2, numerous soft drusen with geographic atrophy; and patient 5, adult-onset foveomacular vitelliform dystrophy) had le-
sions that clinically can resemble various stages of Best vitelliform dystrophy. Given the potential correlation of these particular phenotypes with mutations in the VMD2 gene, it is possible that “vitelliformlike” maculopathies may share a common origin. Patient 1 (Table 1) had a change in exon 6 of the gene, which encodes bestrophin; however, the mutation was different (751C 3 T) from the one previously reported at exon 6 by Petrukhin et al.21 Patients 2 and 3 had a mutation in exon 10 of bestrophin, in a region that has not been reported previously to be affected in patients with maculopathies. Another patient with bull’s-eye maculopathy of unknown cause, patient 4, had a novel mutation in exon 4. It is important to discuss whether these are diseasecausing mutations. The amino acid changes detected in the cases reported here were not found in a control group of similar ethnicity and age comprised of 196 individuals (392 chromosomes).23 As expected, we did find mutations (P297S and E300D) on one allele of two patients with known Best disease23 (data not shown). Two variants, T216I and L567F, were present in three patients diagnosed with ARM without a known family history of the disease. We cannot unequivocally state that the L567F alteration is pathogenic, because Leu567 is not conserved in evolution and because the L567F substitution does not result in change of charge or polarity. Conversely, we failed to detect this alteration in any of the control individuals, and the two patients appeared to share the same haplotype. Furthermore, two paternal half-siblings of patient 3 without maculopathy
Figure 6. Pedigree with autosomal dominant macular dystrophy (family of patient 5). VMD2 ⫽ Best Macular Dystrophy Gene Mutations.
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Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease Table 1. Clinical Data Regarding Five Patients with Variants in the Best (VMD2) Gene Patient No. Age at (Age [yrs], Diagnosis Family Gender) (yrs) History 1 (72, male)
59
No
2 (76, male)
66
No
3 (61, female)
57
4 (77, female)
5 (60, female)
Visual Acuity
Clinical Features in the Right Eye
20/25 both eyes
Clinical Features in the Left Eye
Soft, confluent drusen evolving into RPED, and ultimately into geographic atrophy 20/40 right Numerous soft drusen eye, 20/250 with central area of left eye geographic atrophy
Soft, confluent drusen evolving into RPED
No
20/25 right Small and intermediate eye, 20/32 drusen in both the left eye macula and extramacular area
Soft drusen and central retinal pigment epithelium alterations in addition to extramacular drusen
73
No
20/50 right Subtle annular area of eye, 20/640 retinal pigment left eye epithelial depigmentation
Annular area of retinal pigment epithelial depigmentation in a bull’s-eye pattern
54
Yes
20/32 right Symmetric, slightly eye, 20/20 elevated yellow lesion left eye with central hyperpigmentation consistent with adult-onset foveomacular vitelliform dystrophy, with scattered drusen
Numerous drusen with scattered areas of retinal pigment epithelial alteration
Numerous soft drusen with central area of geographic atrophy
Fluorescein Angiogram Results
Electro-oculogram Results
Hyperfluorescence 3.2/3.4 related to drusen and RPED, no signs of CNVM Hyperfluorescence 3.3/2.5 consistent with staining of the drusen and window defect in areas of geographic atrophy Hyperfluorescence 2.0/2.1 consistent with staining of the drusen and window defects consistent with RPE atrophy Central area of Not available hypofluorescence (patient deceased) with a surrounding ring of hyperfluorescence in the left eye Ring of 1.01/1.5 hyperfluorescence with hypofluorescence centrally and late staining of the central vitelliformlike lesion
CNVM ⫽ choroidal neovascular membrane; RPED ⫽ retinal pigment epithelial detachment.
(one self-reported and one examined), did not have the mutation in the VMD2 gene. Longer follow-up will be needed to confirm the absence of any maculopathy. The second ARM variant, T216I in 1 of 259 subjects, is most likely a disease-causing alteration: Thr216 is located close to the known Best disease mutations, is conserved in evolution from mouse to man, results in a change in polarity, and was not found in the 196 comparison subjects. The two sequence variants found in the cases of bull’s-eye retinopathy (E119Q) and adult-onset foveomacular vitelliform dystrophy (A146K) represent nonconservative substitutions resulting in a gain or loss of charge and are located in evolutionary conserved regions of the protein. In fact, glutamic acid in position 119 is conserved even in Caenorhabditis elegans. These findings indicate that these alterations are likely to be disease-specific mutations. To our knowledge, patient 5 represents the first case description of a patient with adult-onset foveomacular vitelliform dystrophy reported to have a mutation in the VMD2 gene. She had the typical adult vitelliform macular lesion in the right eye19 and several drusen and pigment irregularities in the left eye. Her ocular photographs and fluorescein angiogram were reviewed independently by two retina specialists and diagnosed as adult-onset foveomacular vitelliform dystrophy. Unlike typical Best disease, she was diagnosed at age 54, the vitelliform lesion in the right eye was
smaller than that seen in Best disease, the fellow eye had typical findings of ARM, and her vision was only mildly affected. However, her EOG results were subnormal in both eyes, in the range consistent with Best disease. The phenotype and distribution of disease in this family are consistent with an autosomal dominant pattern dystrophy. Felbor et al25 reported that 18% of patients with adultonset foveomacular vitelliform dystrophy have a mutation in the peripherin/RDS gene, therefore, the mutation in VMD2 reported here represents only the second case where mutation in a gene is associated with this condition. Thus, this case demonstrates the genetic heterogeneity of adultonset foveomacular vitelliform macular dystrophy and underscores observations that one disease can be associated with mutations in more than one gene. Furthermore, our findings raise the possibility that some of these patients may actually have an atypical form of Best disease with late onset. Additional screening of a large series of cases is needed to confirm and expand these findings. Given our limited therapeutic success with AMD to date, disease prevention is becoming an important research priority. Preventive strategies are aimed at the detection and modification of various risk factors associated with the pathogenesis of a disease. With respect to ARM, lifestyle factors, such as smoking,26 may play a role in its pathogenesis. The importance of determining the genetic basis of a
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Ophthalmology Volume 108, Number 11, November 2001 disease could also be viewed in the realm of disease prevention. Depending on penetrance and gene expression, it one day may be possible to identify patients who are in the preclinical phase of disease and genetically susceptible, and offer treatment at an earlier stage. Because some of our patients diagnosed with ARM have mutations in the VMD2 gene, it is possible that this gene is responsible for a small proportion of ARM cases (⬍2%). There is an alternative explanation for our findings. Phenotypically, some fundus features associated with Best disease are similar to those of ARM, especially stages 1 (RPE mottling), 4a (geographic atrophy), 4b (disciform response), and 4c (choroidal neovascularization). Furthermore, the “pseudovitelliform” and “confluent drusen” types of RPE detachments27 clinically can resemble the vitelliform lesions seen in Best disease. Thus, given the phenotypic heterogeneity seen with Best disease, it is possible that the patients with ARM described in this report in fact had a form of Best disease. However, the age at diagnosis of these lesions, the age at onset of visual loss, the morphologic features of the lesions, the prominence of drusen, the lack of family history of visual loss, and the normal EOG results in the two ARM patients who had EOG testing make this possibility unlikely. Identifying the gene associated with a disease can also reveal mechanisms responsible for the pathogenesis of that disease and related conditions. For example, could mutations in the VMD2 gene lead to alteration in proteins, deranged photoreceptors, accumulation of lipofuscin, and damage to RPE cells as seen in Best disease, adult-onset foveomacular vitelliform dystrophy, as well as some forms of ARM?28 Gene identification unlocks the path to discovering why this abnormal material accumulates in the RPE and the associated mechanisms. Our findings related to mutations in the VMD2 gene also demonstrate the association of multiple phenotypes with different mutations in the same gene. Modifier genes may cause different expression of some genes. In addition to the adult vitelliform case reported here, another type of pattern dystrophy has also been seen in the fellow eye of a patient with Best disease29 and in the family members of Best pedigrees.30 The bull’s-eye retinopathy in our series may represent a form of related macular dystrophy,19,20 although work-up could not be completed to derive a definitive diagnosis in this patient because she is deceased. After extensive review of available records from three physicians over several years, no history of chloroquine or hydroxychloroquine use was found in her case, although this cannot be ruled out. One could speculate that patients who carry mutations in this gene have increased susceptibility to these pharmacologic agents. In summary, VMD2 gene mutations may be found in patients with dystrophic forms of maculopathy, other than classic Best disease. Adult-onset foveomacular vitelliform dystrophy is genetically heterogeneous and in some cases may be a form of Best disease with delayed onset, suggesting that these two diseases may be different manifestations of the same disease process. The VMD2 gene does not seem to play a significant role in ARM. However, in a few cases, VMD2 mutations may increase susceptibility to some forms
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of AMD, or a few cases with visual loss and maculopathy diagnosed in middle age as AMD may have some form of Best disease. The associations of VMD2 gene mutations with adult vitelliform dystrophy and, in a few cases, with drusenoid RPE detachments and a bull’s-eye pattern of maculopathy deserve further investigation. Additional genetic studies will improve the existing classification of retinal diseases. Acknowledgments: The authors thank the patients and the referring physicians, without whose cooperation this work would not have been possible.
References 1. Blodi CF, Stone EM. Best’s vitelliform dystrophy. Ophthalmic Pediatr Genet 1990;11:49 –59. 2. Godel V, Chaine G, Regenbogen L, Coscas G. Best’s vitelliform macular dystrophy. Acta Ophthalmol Suppl 1986;175: 1–31. 3. Cross HE, Bard L. Electro-oculography in Best macular dystrophy. Am J Ophthalmol 1974;77:46 –50. 4. Gass JDM. A clinicopathologic study of a peculiar foveomacular dystrophy. Trans Am Ophthalmol Soc 1974;72:139 – 56. 5. Vine AK, Schatz H. Adult-onset foveomacular pigment epithelial dystrophy. Am J Ophthalmol 1980;89:680 –91. 6. Patrinely JR, Lewis RA, Font RI. Foveomacular vitelliform dystrophy, adult type. A clinicopathologic study including electron microscopic observations. Ophthalmology 1985;92: 1712– 8. 7. Seddon JM, Ajani UA, Mitchell BD. Familial aggregation of age-related maculopathy. Am J Ophthalmol 1997;123:199 – 206. 8. Klaver CC, Wolfs RCW, Assink JJM, et al. Genetic risk of age-related maculopathy. Population-based familial aggregation study. Arch Ophthalmol 1998;116:1646 –51. 9. Meyers SM, Greene T, Gutman FA. A twin study of agerelated macular degeneration. Am J Ophthalmol 1995;120: 757– 66. 10. Klein ML, Mauldin WM, Stoumbos VD. Heredity and agerelated macular degeneration. Observations in monozygotic twins. Arch Ophthalmol 1994;112:932–7. 11. Heiba IM, Elston RC, Klein BEK, Klein R. Sibling correlations and segregation analysis of age-related maculopathy: the Beaver Dam Eye Study. Genet Epidemiol 1994;11:51– 67. 12. De La Paz MA, Pericak-Vance MA, Lennon F, et al. Exclusion of TIMP3 as a candidate locus in age-related macular degeneration. Invest Ophthalmol Vis Sci 1997;38:1060 –5. 13. Allikmets R, Shroyer NF, Singh N, et al. Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science 1997;277:1805–7. 14. Stone EM, Webster AR, Vandenburgh K, et al. Allelic variation in ABCR associated with Stargardt disease but not age-related macular degeneration [letter]. Nat Genet 1998;20: 328 –9. 15. Allikmets R. Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium. Am J Hum Genet 2000; 67:487–91. 16. Klein ML, Schultz DW, Edwards A, et al. Age-related macular degeneration. Clinical features in a large family and linkage to chromosome 1q. Arch Ophthalmol 1998;116: 1082– 8.
Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease 17. Bird AC, Bressler NM, Bressler SB, et al. An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol 1995;39: 367–74. 18. Scott IU, Flynn HW Jr, Smiddy WE. Bull’s-eye maculopathy associated with chronic macular hole [letter]. Arch Ophthalmol 1998;116:1116 –7. 19. Gass JDM. Stereoscopic Atlas of Macular Diseases: Diagnosis and Treatment, 4th ed. St. Louis: Mosby-Year Book, 1997; 316 – 8. 20. van den Biesen PR, Deutman AF, Pinckers AJGL. Evolution of benign concentric annular macular dystrophy. Am J Ophthalmol 1985;100:73– 8. 21. Petrukhin K, Koisti MJ, Bakall B, et al. Identification of the gene responsible for Best macular dystrophy. Nat Genet 1998; 19:241–7. 22. Marquardt A, Stohr H, Passmore LA, et al. Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best’s disease). Hum Mol Genet 1998;7:1517–25. 23. Allikmets R, Seddon JM, Bernstein PS, et al. Evaluation of the Best disease gene in patients with age-related macular degen-
24.
25. 26. 27. 28. 29. 30.
eration and other maculopathies. Hum Genet 1999;104:449 – 53. Allikmets R, Singh N, Sun H, et al. A photoreceptor cellspecific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet 1997;15: 236 – 46. Felbor U, Schilling H, Weber BH. Adult vitelliform macular dystrophy is frequently associated with mutations in the peripherin/RDS gene. Hum Mutat 1997;10:301–9. Seddon JM, Willett WC, Speizer FE, Hankinson SE. A prospective study of cigarette smoking and age-related macular degeneration in women. JAMA 1996;276:1141– 6. Hartnett ME, Weiter JJ, Garsd A, Jalkh AE. Classification of retinal pigment epithelial detachments associated with drusen. Graefes Arch Clin Exp Ophthalmol 1992;230:11–9. Kennedy CJ, Rakoczy PE, Constable IJ. Lipofuscin of the retinal pigment epithelium: a review. Eye 1995;9:763–71. Giuffre G, Lodato G. Vitelliform dystrophy and pattern dystrophy of the retinal pigment epithelium: concomitant presence in a family. Br J Ophthalmol 1986;70:526 –32. Gutman I, Walsh JB, Henkind P. Vitelliform macular dystrophy and butterfly-shaped epithelial dystrophy: a continuum? Br J Ophthalmol 1982;66:170 –3.
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Originally received: June 14, 1999. Accepted: June 4, 2001. Manuscript no. 99307. 1 Department of Ophthalmology, Massachusetts Eye Ear Infirmary, Harvard Medical School, Boston, Massachusetts. 2 Epidemiology Unit, Massachusetts Eye Ear Infirmary, Harvard Medical School, Boston, Massachusetts. 3 Department of Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, Utah. 4 Department of Ophthalmology, Columbia University, New York, New York. 5 Department of Human Genetics, Merck Research Laboratories, West Point, Pennsylvania.
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Department of Pathology, Columbia University, New York, New York. Presented as a poster at the American Academy of Ophthalmology annual meeting, Orlando, Florida, October 1999. Supported in part by the National Eye Institute, Bethesda, Maryland (grant nos.: EY11309 [JMS] and EY11600 [PSB]; Research to Prevent Blindness, Inc., New York, New York; an RPB Lew R. Wasserman Merit Award (JMS); an RPB career development award (PSB and RA); the Lions Eye Research Fund, Inc., Northboro, Mass., an unrestricted grant to Moran Eye Center; and The Ruth and Milton Steinbach Fund, New York, NY. The authors have no proprietary interest in the products or devices mentioned herein. Correspondence and reprint requests to Johanna M. Seddon, MD, Department of Ophthalmology, Epidemiology Unit, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114. E-mail: [email protected]. ISSN 0161-6420/01/$–see front matter PII S0161-6420(01)00777-1
Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease Best disease is an autosomal dominant macular dystrophy characterized by the early onset of vitelliform macular lesions. Patients may be asymptomatic or have visual loss at the time of presentation.1 The fundus findings of patients with Best disease can be classified into five stages: no demonstrable pathologic features with abnormal Arden ratios on electro-oculography (EOG) (stage 0), mild retinal pigment epithelial disturbance presenting as window defects on fluorescein angiography (stage 1), vitelliform lesion and scrambled egg appearance (stage 2), pseudohypopyon (stage 3), orange-red atrophic lesion (stage 4a), macular scarring (stage 4b), and neovascularization (stage 4c).2 In Best disease, the results of EOG are abnormal, whereas the electroretinogram results are typically normal.3 Adult-onset foveomacular vitelliform dystrophy is a disorder characterized by the presence of a small, round, yellow subretinal lesion, with a central pigmented spot, in a middle-aged patient.4,5 Drusen may be present with this lesion. On angiography, the central pigmented areas are associated with relative hypofluorescence, whereas the surrounding “vitelliform” material is associated with hyperfluorescence. The EOG Arden ratios in patients with adultonset foveomacular dystrophy are usually normal or minimally subnormal. This disease is typically associated with minimal visual loss. Histologically, these lesions correspond to large amounts of lipofuscin within retinal pigment epithelial (RPE) cells, pigment-laden macrophages in the retina, focal loss of photoreceptors, and RPE cell atrophy.6 There are some phenotypical similarities among Best disease, adult vitelliform maculopathy, and the more common problem of age-related maculopathy (ARM). There is also increasing interest in the hypothesis that ARM may be, in part, a genetic disease. Scientific evidence that ARM has a genetic basis comes from numerous studies. A recent case-control study quantified that macular degeneration is 2.5 times more common among first-degree relatives of case probands, when compared with first-degree relatives of control probands.7 This familial aggregation was confirmed in a population-based study by Klaver et al.8 A few small twin studies have estimated a higher concordance rate between monozygotic twins than dizygotic twins.9,10 Segregation studies suggest that there is a single major gene defect in a large proportion of affected individuals.11 The TIMP3 gene, responsible for Sorsby’s fundus dystrophy, was ruled out as a candidate gene for ARM.12 However, Allikmets et al13 reported that ARM patients, particularly those with dry ARM, had a higher prevalence of mutations in the Stargardt gene (ABCR) when compared with controls. This observation has been investigated in other study populations.14,15 In a recent report analyzing a large family of patients with macular degeneration, the disease locus for this particular family mapped to chromosome 1q25-q31 between markers D1S466 and D1S413.16 Age-related maculopathy has a myriad of manifestations that may be pathogenetically distinct. Clinical signs of ARM include: drusen, RPE hyperplasia or depigmentation (collectively known as early ARM), and geographic atrophy, RPE detachment, and choroidal neovascularization (collec-
tively known as advanced ARM or age-related macular degeneration [AMD]).17 Bull’s-eye maculopathy refers to the ophthalmoscopic finding of depigmentation of central retinal pigment epithelium surrounded by a relatively normal area, giving a bull’seye appearance to the macula. This phenotype is associated with a number of macular diseases, including chloroquine and hydroxychloroquine maculopathies, cone dystrophy as well as Stargardt disease, benign concentric annular dystrophy, and a variety of other macular dystrophies.18 –20 The gene causing Best macular dystrophy (VMD2) has recently been cloned and is located on chromosome 11 at 11q13.21 Petrukhin et al21 identified five independent disease-specific mutations in five families with Best disease. These mutations were associated with nucleic acid changes at exons 2, 4, 6, and 8. Marquardt et al22 also reported mutations in this gene (VMD2) in patients with Best disease. The Best gene is heavily expressed in the retina, specifically in the RPE. Because the gene associated with Best disease is a good candidate for other maculopathies, we screened the DNA of patients and controls in our databases for the presence of variants in this gene. We recently reported that novel mutations in the VMD2 gene were found in some of our patients with Best macular dystrophy as well as other maculopathies not diagnosed with classic Best disease.23 In the present study, we describe in detail the clinical features and phenotypes of the patients diagnosed with maculopathies other than Best disease, as well as some of their family members, and list their variant sequences in the VMD2 gene. Results suggest that different mutations in a single gene can result in a variety of phenotypes and support the likelihood that emerging new genetic information will modify definitions for the clinical diagnosis of various maculopathies.
Materials and Methods The Family Macular Degeneration Study is a National Institutes of Health-supported, Massachusetts Eye and Ear Infirmary Human Subject Committee-approved study being performed to determine the genetic basis of macular degeneration. As part of this study, patients are asked questions about their family members, and a complete pedigree is constructed. All participants consent to a thorough ocular evaluation with fundus photography, which may also include diagnostic testing when appropriate (fluorescein angiography, electrophysiologic testing, or both). They also provide blood and buccal swab sources of DNA to be used for genetic testing. From the Massachusetts Eye and Ear Infirmary site, we screened the DNA samples from 188 patients with ARM who were classified in a standardized manner as previously described [Seddon et al. Invest Ophthalmol Vis Sci 38(Suppl):S675, 1997]: 155 (82%) with the “dry” type of the disease, including extensive intermediate drusen, large drusen, or geographic atrophy, and the remaining 33 (18%) with the “wet,” or exudative form with fluorescein angiographic evidence of choroidal neovascularization, and 28 patients with maculopathies other than classic Best disease. These 28 cases included myopic retinal degeneration (n ⫽ 1), macular RPE changes or RPE detachment of uncertain cause in young patients (n ⫽ 2), cone dystrophy (n ⫽ 4), multifocal RPE detachment consistent with a diagnosis of multifocal Best disease
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Figure 1. Patient 1. A, Drusenoid-type of retinal pigment epithelial detachment, right eye. B, Geographic atrophy corresponding to previous area of retinal pigment epithelial detachment, right eye.
Figure 2. Patient 2. Numerous soft drusen with central area of geographic atrophy, right eye.
Figure 3. Patient 3. Soft drusen and central area of retinal pigment epithelial alteration associated with extramacular drusen, left eye.
Figure 4. Patient 4. Few drusen associated with a ring like area of retinal pigment epithelial depigmentation, resembling a bull’s eye, left eye.
Figure 5. Patient 5. A, Symmetric, slightly elevated yellow lesion with central hyperpigmentation consistent with adult-onset foveomacular vitelliform dystrophy. Fine drusen are also present, right eye. B, Fluorescein angiogram demonstrating the ring of hyperfluorescence with hypofluorescence centrally and late staining of the central vitelliformlike lesion, right eye.
Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease (n ⫽ 1), atypical retinitis pigmentosa (n ⫽ 1), Stargardt disease (n ⫽ 2), bull’s-eye maculopathy (n ⫽ 5), pattern dystrophy (n ⫽ 5), early onset of drusen (n ⫽ 1), RPE changes with family history of pattern dystrophy (n ⫽ 1), annular pigment dystrophy (n ⫽ 2), and adult-onset foveomacular vitelliform dystrophy (n ⫽ 3). DNA samples from an additional 71 patients with ARM from the Moran Eye Center in Utah were also screened. All of these subjects signed consent forms to participate in the research protocol. They were classified in the same manner as the ARM cases at the Massachusetts Eye and Ear Infirmary, and 10 cases (14%) had the exudative form of the disease. The control group consisted of 196 white subjects of similar ethnicity and age range as the cases (ages 65 years and older)who were examined by two of the authors (JMS and PSB) and found not to have ARM or any other maculopathy. All 11 exons of the VMD2 gene, as well as 5⬘ and 3⬘ untranslated regions, were screened for variants using single-strand conformation polymorphism analysis and direct sequencing of variants in all patients and controls as described previously.21,24 Five patients were identified with amino acid-changing variants in the VMD2 gene, and none of these variants were found in the 196 controls (392 chromosomes). All other variants found in the VMD2 gene, including several common alleles, have been described elsewhere.23 The purpose of this report was to describe the clinical features of these five cases and some of their family members.
Results Case Reports Patient 1. A 72-year-old male noted mild gradual visual deterioration over a 13-year period. The patient reported no family history of eye problems or visual loss. In February 1995, his visual acuity was recorded as 20/25 in each eye. His anterior segment examination results were within normal limits. Funduscopic examination revealed large confluent drusen and hypopigmentation and clumping of RPE in both eyes. There was no evidence of basal laminar drusen in either eye. Fluorescein angiography demonstrated staining of drusen, but no evidence of choroidal neovascularization. The patient was seen again in July 1996. At that time, his visual acuity was 20/32 in the right eye and 20/40 in the left eye. Examination of the fundus revealed bilateral confluent drusen and RPE detachments (Fig 1A, right eye).5 Fluorescein angiography demonstrated staining of drusen and pooling of dye in the area of the RPE detachments. There was no evidence of choroidal neovascularization. When the patient was examined again in June 1998, visual acuity was 20/25 in the right eye and 20/50 in the left eye. On ophthalmoscopic examination, geographic atrophy in the right eye and large drusemoid-type RPE detachment in the left eye were present (Fig 1B, right eye). Electro-oculography revealed normal Arden ratios of 3.2 in the right eye and 3.4 in the left eye. A 751C 3 T variant was found in exon 6 of the VMD2 gene, resulting in a T216I mutation. Patient 2. A 76-year-old male described gradual visual loss over a 10-year period. The patient reported no family history of eye problems or visual loss. In September 1993, the patient’s visual acuity was 20/40 in the right eye and finger counting in the left eye. He was pseudophakic in the right eye and had a moderate cataract in the left eye. He was noted to have numerous soft confluent drusen in the right eye and numerous soft drusen with geographic atrophy in the left eye. There were no extramacular drusen or peripheral reticular pigmentary changes. The patient underwent uncomplicated cataract extraction with intraocular lens implantation in the left eye a few months after initial assessment. His visual acuity during his most recent examination (September 1998) was 20/40 in the right eye and 20/250 in the left eye. His
funduscopic examination revealed numerous soft drusen with some degree of confluence and geographic atrophy in both eyes (Fig 2; right eye). Fluorescein angiography revealed hyperfluorescence of the drusen and late frame hyperfluorescence and staining associated with the area of geographic atrophy. Electro-oculography revealed Arden ratios of 3.3 in the right eye and 2.5 in the left eye. A 1803C 3 T (L567F) variant was found in exon 10 of the VMD2 gene. Patient 3. A 61-year-old female noted a mild gradual visual deterioration in the right eye over a 4-year period. The patient reported no family history of eye problems or visual loss. Her visual acuity was 20/25 in each eye. Fundus examination demonstrated small and intermediate drusen in the right eye and large soft drusen in the left eye, consistent with dry ARM, as well as extramacular drusen (Fig 3, left eye). No reticular pigmentary changes were noted in the periphery of either eye. Fluorescein angiography revealed hyperfluorescence of the drusen and window defects consistent with the areas of RPE alteration. Three years later, visual acuity was 20/25 in the right eye and 20/32 in the left eye, and funduscopic examination and fluorescein angiogram were unchanged. The same variant as patient 2, 1803C 3 T (L567F) was found in exon 10 of the VMD2 gene in this patient. The EOG results were normal and revealed Arden ratios of 2.0 in the right eye and 2.1 in the left eye. Two half-siblings (same father) also underwent genotyping: a male age 46 and a female age 50. The female was examined and had no evidence of maculopathy and the male reported no eye disease. Neither relative had the mutation in the VMD2 gene. Patient 4. A 77-year-old female noted a gradual decline in her vision over a 4-year period. The patient reported no family history of eye problems or visual loss. She had a history of systemic lupus erythematosus and had been taking medication of uncertain type in the past 10 years. A thorough search of her past records revealed no history of chloroquine or hydroxychloroquine therapy. Her visual acuity was 20/50 in the right eye and 20/640 in the left eye. Funduscopic examination revealed early annular RPE depigmentation in the right eye and an annular area of retinal pigment depigmentation, consistent with a bull’s-eye maculopathy in the left eye (Fig 4). Reticular pigmentary changes were noted in both eyes, without signs of extramacular drusen. Angiography revealed a central area of hypofluorescence surrounded by an annular ring of hyperfluorescence. There was no evidence of either a “dark choroid” or other lesions on angiography. Electro-oculography could not be performed because the patient is deceased. A 459G 3 C (E119Q) mutation was detected in exon 4 of the VMD2 gene. Patient 5. A 60-year-old female noted mild visual deterioration in the right eye over a 6-year period. Her visual acuity was 20/32 in the right eye and 20/20 in the left eye. Her ophthalmoscopic examination revealed a small RPE detachment with central hyperpigmentation in the right eye consistent with the diagnosis of adult-onset foveomacular vitelliform dystrophy (Fig 5A). Mottling of the RPE and fine drusen were also seen. The photographs and fluorescein angiograms were reviewed independently by two other retina specialists who agreed with the clinical diagnosis of adultonset vitelliform macular dystrophy. Mild reticular pigmentary changes were noted in the periphery of the right eye, without extramacular drusen. Fluorescein angiography in the right eye revealed relative hypofluorescence corresponding to the central area of hyperpigmentation and late frame staining in the area of deposition of vitelliform material (Fig 5B). Electro-oculography revealed abnormal Arden ratios of 1.01 in the right eye and 1.5 in the left eye. Sequencing revealed a 540GC 3 AA (A146K) mutation in exon 4 of the VMD2 gene in this patient. This patient informed us that her deceased brother (Fig 6) had macular degeneration, which was diagnosed at age 67. After obtaining consent from the family, we obtained his ocular photo-
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Ophthalmology Volume 108, Number 11, November 2001 graphs and fluorescein angiogram, which demonstrated that he had maculopathy in both eyes consisting of a ring of yellow deposits in the right eye and central pigment alteration in the left eye. Angiographically, the lesion in his left eye had a nonfluorescent center surrounded by a ring of hyperfluorescence, which are features resembling adult-onset foveomacular vitelliform dystrophy. The mother in this family had decreased central vision and a history of maculopathy. The three sons of the proband (ages 46, 52, and 53) underwent genotyping, and two were found to have the same variant in the VMD2 gene and one (age 52) did not. This latter son was examined and photographed and did not have any evidence of maculopathy. Thus far, we have been unable to arrange an ocular examination, photography, or EOG for the two sons with the variants in the Best gene.
Discussion Four unique variants were detected in the VMD2 (Best) gene in 5 patients with maculopathies: 3 of 259 subjects (approximately 1%) with age-related maculopathy, and 2 of 28 other subjects (approximately 7%) with maculopathies not diagnosed as classic Best disease. These two latter cases with genetic variants in VMD2 included one of three subjects with adult-onset foveomacular vitelliform maculopathy and one of five subjects with bull’s-eye maculopathy. It is interesting to note that three of these five patients (patient 1, confluent drusen type of RPE detachment; patient 2, numerous soft drusen with geographic atrophy; and patient 5, adult-onset foveomacular vitelliform dystrophy) had le-
sions that clinically can resemble various stages of Best vitelliform dystrophy. Given the potential correlation of these particular phenotypes with mutations in the VMD2 gene, it is possible that “vitelliformlike” maculopathies may share a common origin. Patient 1 (Table 1) had a change in exon 6 of the gene, which encodes bestrophin; however, the mutation was different (751C 3 T) from the one previously reported at exon 6 by Petrukhin et al.21 Patients 2 and 3 had a mutation in exon 10 of bestrophin, in a region that has not been reported previously to be affected in patients with maculopathies. Another patient with bull’s-eye maculopathy of unknown cause, patient 4, had a novel mutation in exon 4. It is important to discuss whether these are diseasecausing mutations. The amino acid changes detected in the cases reported here were not found in a control group of similar ethnicity and age comprised of 196 individuals (392 chromosomes).23 As expected, we did find mutations (P297S and E300D) on one allele of two patients with known Best disease23 (data not shown). Two variants, T216I and L567F, were present in three patients diagnosed with ARM without a known family history of the disease. We cannot unequivocally state that the L567F alteration is pathogenic, because Leu567 is not conserved in evolution and because the L567F substitution does not result in change of charge or polarity. Conversely, we failed to detect this alteration in any of the control individuals, and the two patients appeared to share the same haplotype. Furthermore, two paternal half-siblings of patient 3 without maculopathy
Figure 6. Pedigree with autosomal dominant macular dystrophy (family of patient 5). VMD2 ⫽ Best Macular Dystrophy Gene Mutations.
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Seddon et al 䡠 Best Macular Dystrophy Gene Mutations in Maculopathies Other Than Classic Best Disease Table 1. Clinical Data Regarding Five Patients with Variants in the Best (VMD2) Gene Patient No. Age at (Age [yrs], Diagnosis Family Gender) (yrs) History 1 (72, male)
59
No
2 (76, male)
66
No
3 (61, female)
57
4 (77, female)
5 (60, female)
Visual Acuity
Clinical Features in the Right Eye
20/25 both eyes
Clinical Features in the Left Eye
Soft, confluent drusen evolving into RPED, and ultimately into geographic atrophy 20/40 right Numerous soft drusen eye, 20/250 with central area of left eye geographic atrophy
Soft, confluent drusen evolving into RPED
No
20/25 right Small and intermediate eye, 20/32 drusen in both the left eye macula and extramacular area
Soft drusen and central retinal pigment epithelium alterations in addition to extramacular drusen
73
No
20/50 right Subtle annular area of eye, 20/640 retinal pigment left eye epithelial depigmentation
Annular area of retinal pigment epithelial depigmentation in a bull’s-eye pattern
54
Yes
20/32 right Symmetric, slightly eye, 20/20 elevated yellow lesion left eye with central hyperpigmentation consistent with adult-onset foveomacular vitelliform dystrophy, with scattered drusen
Numerous drusen with scattered areas of retinal pigment epithelial alteration
Numerous soft drusen with central area of geographic atrophy
Fluorescein Angiogram Results
Electro-oculogram Results
Hyperfluorescence 3.2/3.4 related to drusen and RPED, no signs of CNVM Hyperfluorescence 3.3/2.5 consistent with staining of the drusen and window defect in areas of geographic atrophy Hyperfluorescence 2.0/2.1 consistent with staining of the drusen and window defects consistent with RPE atrophy Central area of Not available hypofluorescence (patient deceased) with a surrounding ring of hyperfluorescence in the left eye Ring of 1.01/1.5 hyperfluorescence with hypofluorescence centrally and late staining of the central vitelliformlike lesion
CNVM ⫽ choroidal neovascular membrane; RPED ⫽ retinal pigment epithelial detachment.
(one self-reported and one examined), did not have the mutation in the VMD2 gene. Longer follow-up will be needed to confirm the absence of any maculopathy. The second ARM variant, T216I in 1 of 259 subjects, is most likely a disease-causing alteration: Thr216 is located close to the known Best disease mutations, is conserved in evolution from mouse to man, results in a change in polarity, and was not found in the 196 comparison subjects. The two sequence variants found in the cases of bull’s-eye retinopathy (E119Q) and adult-onset foveomacular vitelliform dystrophy (A146K) represent nonconservative substitutions resulting in a gain or loss of charge and are located in evolutionary conserved regions of the protein. In fact, glutamic acid in position 119 is conserved even in Caenorhabditis elegans. These findings indicate that these alterations are likely to be disease-specific mutations. To our knowledge, patient 5 represents the first case description of a patient with adult-onset foveomacular vitelliform dystrophy reported to have a mutation in the VMD2 gene. She had the typical adult vitelliform macular lesion in the right eye19 and several drusen and pigment irregularities in the left eye. Her ocular photographs and fluorescein angiogram were reviewed independently by two retina specialists and diagnosed as adult-onset foveomacular vitelliform dystrophy. Unlike typical Best disease, she was diagnosed at age 54, the vitelliform lesion in the right eye was
smaller than that seen in Best disease, the fellow eye had typical findings of ARM, and her vision was only mildly affected. However, her EOG results were subnormal in both eyes, in the range consistent with Best disease. The phenotype and distribution of disease in this family are consistent with an autosomal dominant pattern dystrophy. Felbor et al25 reported that 18% of patients with adultonset foveomacular vitelliform dystrophy have a mutation in the peripherin/RDS gene, therefore, the mutation in VMD2 reported here represents only the second case where mutation in a gene is associated with this condition. Thus, this case demonstrates the genetic heterogeneity of adultonset foveomacular vitelliform macular dystrophy and underscores observations that one disease can be associated with mutations in more than one gene. Furthermore, our findings raise the possibility that some of these patients may actually have an atypical form of Best disease with late onset. Additional screening of a large series of cases is needed to confirm and expand these findings. Given our limited therapeutic success with AMD to date, disease prevention is becoming an important research priority. Preventive strategies are aimed at the detection and modification of various risk factors associated with the pathogenesis of a disease. With respect to ARM, lifestyle factors, such as smoking,26 may play a role in its pathogenesis. The importance of determining the genetic basis of a
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Ophthalmology Volume 108, Number 11, November 2001 disease could also be viewed in the realm of disease prevention. Depending on penetrance and gene expression, it one day may be possible to identify patients who are in the preclinical phase of disease and genetically susceptible, and offer treatment at an earlier stage. Because some of our patients diagnosed with ARM have mutations in the VMD2 gene, it is possible that this gene is responsible for a small proportion of ARM cases (⬍2%). There is an alternative explanation for our findings. Phenotypically, some fundus features associated with Best disease are similar to those of ARM, especially stages 1 (RPE mottling), 4a (geographic atrophy), 4b (disciform response), and 4c (choroidal neovascularization). Furthermore, the “pseudovitelliform” and “confluent drusen” types of RPE detachments27 clinically can resemble the vitelliform lesions seen in Best disease. Thus, given the phenotypic heterogeneity seen with Best disease, it is possible that the patients with ARM described in this report in fact had a form of Best disease. However, the age at diagnosis of these lesions, the age at onset of visual loss, the morphologic features of the lesions, the prominence of drusen, the lack of family history of visual loss, and the normal EOG results in the two ARM patients who had EOG testing make this possibility unlikely. Identifying the gene associated with a disease can also reveal mechanisms responsible for the pathogenesis of that disease and related conditions. For example, could mutations in the VMD2 gene lead to alteration in proteins, deranged photoreceptors, accumulation of lipofuscin, and damage to RPE cells as seen in Best disease, adult-onset foveomacular vitelliform dystrophy, as well as some forms of ARM?28 Gene identification unlocks the path to discovering why this abnormal material accumulates in the RPE and the associated mechanisms. Our findings related to mutations in the VMD2 gene also demonstrate the association of multiple phenotypes with different mutations in the same gene. Modifier genes may cause different expression of some genes. In addition to the adult vitelliform case reported here, another type of pattern dystrophy has also been seen in the fellow eye of a patient with Best disease29 and in the family members of Best pedigrees.30 The bull’s-eye retinopathy in our series may represent a form of related macular dystrophy,19,20 although work-up could not be completed to derive a definitive diagnosis in this patient because she is deceased. After extensive review of available records from three physicians over several years, no history of chloroquine or hydroxychloroquine use was found in her case, although this cannot be ruled out. One could speculate that patients who carry mutations in this gene have increased susceptibility to these pharmacologic agents. In summary, VMD2 gene mutations may be found in patients with dystrophic forms of maculopathy, other than classic Best disease. Adult-onset foveomacular vitelliform dystrophy is genetically heterogeneous and in some cases may be a form of Best disease with delayed onset, suggesting that these two diseases may be different manifestations of the same disease process. The VMD2 gene does not seem to play a significant role in ARM. However, in a few cases, VMD2 mutations may increase susceptibility to some forms
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of AMD, or a few cases with visual loss and maculopathy diagnosed in middle age as AMD may have some form of Best disease. The associations of VMD2 gene mutations with adult vitelliform dystrophy and, in a few cases, with drusenoid RPE detachments and a bull’s-eye pattern of maculopathy deserve further investigation. Additional genetic studies will improve the existing classification of retinal diseases. Acknowledgments: The authors thank the patients and the referring physicians, without whose cooperation this work would not have been possible.
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