Review article Genomic Medicine W. Gregory Feero, M.D., Ph.D., and Alan E. Guttmacher, M.D., Editors Genomics and the Eye Val C. Sheffield, M.D., Ph.D., and Edwin M. Stone, M.D., Ph.D. From the Departments of Pediatrics (V.C.S.) and Ophthalmology and Visual Sciences (V.C.S., E.M.S.), Howard Hughes Medical Institute, University of Iowa Carver College of Medicine, Iowa City. Address reprint requests to Dr. Stone at the University of Iowa Institute for Vision Research, 375 Newton Rd., Iowa City, IA 52242, or at [email protected] uiowa.edu. This article (10.1056/NEJMra1012354) was updated on May 19, 2011, at NEJM.org. N Engl J Med 2011;364:1932-42. Copyright © 2011 Massachusetts Medical Society. T he eye has had a pivotal role in the evolution of human genomics. At least 90% of the genes in the human genome are expressed in one or more of the eye’s many tissues and cell types at some point during a person’s life. Consistent with this impressive genomic footprint is the observation that about a third of entries in the Online Mendelian Inheritance in Man database for which a clinical synopsis is provided include a term that refers to the structure or function of the eye.1 Moreover, the phenotypic effects of even small genetic variations are made readily apparent by the many layers of amplification in the human visual system. For example, a single-nucleotide change in PAX6 can cause an anatomic abnormality of the macula less than a millimeter in diameter that results in noticeably reduced visual acuity and nystagmus.2 The heritable inability to correctly perceive the color green, known as Daltonism (after the English chemist John Dalton, who himself was affected), was the first human trait mapped to the X chromosome.3 (See Fig. 1 for a timeline of historic discoveries.) The Coppock cataract was the first human trait mapped to an autosome,4 and Leber’s hereditary optic neuropathy was the first human disease shown to be caused by a mutation in mitochondrial DNA.5 More recently, age-related macular degeneration (AMD) and glaucoma6,7 — two common causes of human blindness — have been shown to be largely genetic, as has Fuchs’ endothelial dystrophy,8 the most common cause of corneal transplantation in developed countries. Here, we review discoveries in mendelian and complex ophthalmic disorders and their implications for genetic testing and therapeutic intervention. Gene t ic Te s t ing The modern era of molecular ophthalmology began in 1985, with the discovery of the retinoblastoma gene.9 Since then, hundreds of other genes that are responsible for a wide variety of important diseases have been discovered, including those associated with AMD, glaucoma, congenital cataract, syndromic and nonsyndromic forms of photoreceptor degeneration, and multiple macular dystrophies, corneal dystrophies, vitreoretinopathies, and optic neuropathies. The discovery of each new genetic cause of disease affords the possibility of using molecular investigation of DNA samples collected from individual patients as an adjunct to clinical diagnosis, prognosis, and counseling. In addition, persons who are found to carry known disease-causing mutations can be enrolled in clinical trials of new therapies or carefully studied with a variety of clinical instruments to fully explore the behavior of their disease over time.10,11 Samples from patients who lack mutations in known disease-causing genes can also serve as a valuable resource for scientists who seek to find additional disease-causing genes. A major challenge in using this emerging genetic information in the clinical 1932 n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. genomic medicine domain is the gap that exists between the amount of information that is needed to convincingly demonstrate a pathogenic role of a given gene in a group of research subjects and the amount of information that is needed to reliably assert that a given genetic variation is responsible for a disease in an individual patient. Some of the factors that are responsible for this gap include the large number of non–phenotype-altering variations scattered throughout many genes, the wide variety of types of true disease-causing mutations 460–322 B.C.E. Hippocrates and Aristotle study familial transmission of ocular traits 1865 Mendel experiments with plant hybridization 1869 Leber describes congenital amaurosis 1800s 1900s (e.g., missense, nonsense, splice-site, promoterinactivation, and copy-number variation) (see Glossary), the genetic differences among different ethnic groups, the genetic heterogeneity of many phenotypes (e.g., retinitis pigmentosa can be caused by a mutation in any one of more than 40 genes), and the clinical variation among patients with similar genotypes. Because all genetic variations are not equally likely to cause disease, some investigators have suggested methods for taking this uncertainty 2011 Ranibizumab and bevacizumab are shown to be essentially equivalent in neovascular AMD 1911 Thomas Morgan identifies the chromosome as the physical repository of genetic material 1953 Structure of DNA is deduced Color blindness is the first human trait mapped to the X chromosome 1997 First human glaucoma gene, MYOC, is identified 1963 Coppock cataract is the first human trait mapped to an autosome 1917 Ophthalmology is the first medical specialty to create its own assessment board 1910s 1920s 1930s 1847 Charles Babbage invents the ophthalmoscope Stargardt’s disease gene, ABCA4, is identified 1971 Folkman recognizes therapeutic implications of tumor angiogenesis factor 1940s 1950s 1960s 1970s 1980s 1990s 2000s 2010s 1983 Polymerase chain reaction is introduced 1985 First human cancer gene, retinoblastoma, is cloned 1987 Rhodopsin is the first gene associated with a retinal degenerative disease 1989 Leber’s hereditary optic neuropathy is the first human disease shown to be caused by a mutation of mitochondrial DNA 2000 Gene therapy for canine blindness caused by Leber’s congenital amaurosis is successful 2004 Bevacizumab is approved for use in colon cancer 2005 AMD is the first eye disease to yield a positive genomewide association 2006 Off-label bevacizumab is shown to be effective in neovascular AMD Ranibizumab is approved for use in neovascular AMD 2007 First gene-replacement therapy for a human eye disease, Leber’s congenital amaurosis, is successful Figure 1. Timeline of Landmarks in Ophthalmic Genetics. AMD denotes age-related macular degeneration. COLOR FIGURE Draft 5 Author Fig # 5/02/11 Stone 1 Title n engl j med 364;20 nejm.org may 19, 2011 1933 ME DE Artist Phimister Knoper The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. AUTHOR PLEASE NOTE: Figure has been redrawn and type has been reset Copyright © 2011 Massachusetts Medical Society. All rights reserved. Please check carefully Issue date 5/19/11 The n e w e ng l a n d j o u r na l of m e dic i n e Glossary Allele: One of two or more versions of a genetic sequence at a particular location in the genome. Autosome: All the chromosomes except for the sex chromosomes and the mitochondrial chromosome. Chaperone complex: An oligomeric protein that assists in the folding, unfolding, assembly, or disassembly of other macromolecular structures without being permanently incorporated into the assisted structures. Codon: A three-nucleotide sequence of DNA or RNA that specifies a single amino acid. Copy-number variation: Variation from one person to the next in the number of copies of a particular gene or DNA sequence. The full extent to which copy-number variation contributes to human disease is not yet known. De novo mutation: Any DNA sequence change that occurs during replication, such as a heritable gene alteration occurring in a family for the first time as a result of a DNA sequence change in a germ cell or fertilized egg. Genomewide association study: An approach used in genetics research to look for associations between typically hundreds of thousands of specific genetic variations (most commonly single-nucleotide polymorphisms) and particular diseases. Linkage analysis: An approach to the discovery of the genetic basis of a disease that correlates the pattern of disease inheritance within families with specific alleles of genetic markers of known location. Locus: The specific chromosomal location of a gene or other DNA sequence of interest. Loss-of-function mutation: A mutation that decreases the production or function of a protein (or does both). Missense mutation: The alteration of a single DNA nucleotide so that the resulting codon specifies a different amino acid. Nonsense mutation: The alteration of a single DNA nucleotide so that the resulting codon signals a termination of translation, thus leading to truncation of the encoded protein. Penetrance: The likelihood that a person carrying a particular genetic variant will have a detectably altered phenotype. Population attributable risk: The difference in the rate of disease between a population that is exposed to a given factor and one that is not. The population attributable risks of individual factors that contribute to a single clinical entity, such as age-related macular degeneration, often total more than 100% because the disease in a specific patient may be caused by a combination of factors that are counted more than once when individual population attributable risks are summed. Promoter-inactivation mutation: A genetic variation in the promoter of an otherwise normal gene that results in a dramatic reduction in gene expression. Single-nucleotide polymorphism: A single-nucleotide variation in a genetic sequence, a common form of variation in the human genome. Splice-site mutation: A sequence variation at or near an intron–exon boundary that perturbs normal splicing of the adjacent intron. into account in a standardized fashion when interpreting the results of genetic testing.12 The advent of whole-exome sequencing as a diagnostic tool accentuates the need for this type of probabilistic interpretation, because every person carries several recessive disease-causing mutations that would be incidental and medically irrelevant to any disease that they might have in their lifetimes. For example, approximately 1 in 30 Europeans is heterozygous for the deletion of codon 508 in the gene that is associated with cystic fibrosis.13 The ability to distinguish between newly encountered benign variants and those that might confer risk is central to the task of interpreting genetic data, especially those generated by large data sets, such as the whole genome of an individual. As in all of medicine, a genetic test result is more likely to be meaningful when it is accom1934 panied by a robust pretest hypothesis. Thus, the growth of molecular ophthalmology has increased the need for experienced clinicians who can place the observed genetic variations in the correct clinical context. Mendel i a n Disor der s According to the World Health Organization, the most common causes of blindness across the globe are cataracts, glaucoma, AMD, corneal opacity, diabetic retinopathy, infections, and parasitic diseases.14 Genetic factors play a role in many of these conditions, sometimes in the form of relatively rare, high-penetrance monogenic diseases and sometimes in the form of more common conditions caused by the complex interplay of multiple genes and the environment. From the n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. genomic medicine many known monogenic eye disorders, we have selected three to illustrate the wide variety of pathophysiological mechanisms involved in human blindness. Mutation in ABCA4 — identified by Allikmets in 1997 as a cause of Stargardt’s disease15 — is one of the most important causes of monogenic retinal disease in humans. ABCA4 is an enzyme that flips a retinoid intermediate of the visual cycle known as N-retinylidene-phosphatidylethanolamine (N-retPE) from the inner leaflet to the outer leaflet of the photoreceptor outer segment disk membrane16 (Fig. 2, and interactive graphic, available with the full text of this article at NEJM .org). Mutations in ABCA4 result in an intradiscal accumulation of N-retPE, which in turn leads to the formation of a toxic, insoluble bisretinoid known as A2E. Variations in ABCA4 are responsible for more than 95% of cases of Stargardt’s disease, 30% of cases of cone–rod dystrophy, and 8% of cases of autosomal recessive retinitis pigmentosa.17 This range of phenotypes results from the interplay of at least three factors: the degree of residual enzymatic function associated with a given genotype, the fact that cones are more readily harmed than rods by the accumulation of A2E, and the fact that injury to the retinal pigment epithelium results in secondary injury to both rods and cones.10 ABCA4 mutations with a relatively mild effect result in the accumulation of A2E within and beneath the retinal pigment epithelium, those with an intermediate effect result in a direct injury to photoreceptors that is somewhat cone-selective, and those with the most severe effect result in injury to both cone and rod photoreceptors.17 A second example, mutation in MYOC in autosomal dominant juvenile-onset primary open-angle glaucoma, involves mistrafficking of a normally secreted trabecular meshwork protein to the peroxisome. Linkage analysis of several large families mapped the causal mutations to the long arm of chromosome 1,18 and further genetic dissection of this locus revealed mutations in MYOC as the cause of the disease.19 Certain missense mutations are associated with very high intraocular pressures and early onset of vision loss, whereas a nonsense mutation at codon 368 is associated with milder disease and a later onset,20 an unexpected finding because nonsense mutations typically have a more severe effect on protein integrity than do missense mutations. It was later discov- ered that the missense mutations in MYOC cause the myocilin protein to misfold, with consequent unmasking of an otherwise cryptic signal that targets myocilin to the peroxisome.21 The resulting intracellular retention of myocilin causes injury to the cells that make up the trabecular meshwork, which in turn reduces the outflow of aqueous humor. The elevated intraocular pressure resulting from this reduced outflow causes injury to the optic nerve. MYOC mutations have been shown to be involved in approximately 4% of all cases of primary open-angle glaucoma, including adult-onset disease.19 A third example illustrates the phenomenon of genetic heterogeneity: mutations in at least 14 genes cause a clinical syndrome known as the Bardet–Biedl syndrome (BBS). BBS is a pleiotropic autosomal recessive disorder that is characterized by the combination of retinitis pigmentosa, obesity, polydactyly, congenital heart defects, renal abnormalities, hypogenitalism, cognitive impairment, and an increased incidence of hypertension and diabetes mellitus.22 Patients with BBS present with progressive photoreceptor degeneration and are usually blind by the third decade of life. Studies of animal models have shown that proteins that are affected by mutations causing BBS are components of cilia or serve in intraflagellar or intracellular transport.23 The fact that the mutation of multiple different genes can be associated with a single pleiotropic phenotype has now been largely explained by the discovery of two BBS protein complexes: the BBSome (consisting of seven BBS proteins), which plays a role in intra­ flagellar transport; and a chaperone complex (con­ sisting of three BBS proteins), which is required for BBSome assembly (Fig. 3). An interactive graphic regarding ABCA4 mutations is available at NEJM.org C ompl e x Disor der s Disorders with complex inheritance result from the interaction of multiple genetic loci and environmental factors such that a mendelian inheritance pattern is not observed. As a result, an allele contributing to a complex disease has a much lower penetrance than an allele involved in a single-gene disorder. This fact affects both the manner in which investigators identify such alleles and the way in which the presence or absence of such alleles has an effect on the care and counseling of patients and their families. As a general rule, linkage analysis of affected families has been n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. 1935 The n e w e ng l a n d j o u r na l more successful in identifying disease-causing alleles in single-gene disorders (e.g., those described in the previous section), whereas genomewide or candidate association studies have been more successful in identifying factors involved in complex diseases, such as AMD, glaucoma, and Fuchs’ endothelial dystrophy. In a counseling context, alleles of single-gene disorders can often be reasonably said to cause disease, whereas alleles that are involved in complex diseases are more commonly said to increase the risk of disease. Three of the most common causes of blindness — AMD, glaucoma, and Fuchs’ endothelial corneal dystrophy — have both genetic heterogeneity and genetic complexity, and genomewide association studies have recently revealed clues to the pathogenicity of all three disorders. AMD is the leading cause of blindness in developed countries. As the name implies, the disorder usually affects persons over the age of 60 years and results in loss of macular (central) vision (Fig. 4). The prevalence of AMD increases with age, and more than 30% of persons over the age of 75 years will have some manifestation of the disease.6 Initial attempts to identify AMD loci involved screening genes that were known to cause monogenic macular disease in patients with AMD and in ethnically matched control subjects.24 Later, the development of cost-effective high-throughput genotyping made genomewide association studies possible. Of all diseases studied in this manner, AMD has been one of the most successful in that it has yielded loci that contribute a high relative risk. For example, persons who carry a certain variant of the gene encoding complement factor H (CFH)25-28 have a relative risk of AMD that is more than 2.7 times that of persons without this variant.25,26 A variant at chromosome 10q26, in the vicinity of three genes (ARMS2, HTRA1, and PLEKHA1), is also strongly associated with AMD.29-31 In all, more than a dozen genes have been linked to AMD.6 Although these studies are important to the further understanding of the pathophysiological mechanisms of AMD and may aid in the development of new therapies, clinical testing for AMD-associated polymorphisms is of little value in the clinical management of AMD at this time. AMD will develop in only about a third of persons with the highestrisk CFH genotype by the age of 70 years. Thus, unless and until a safe and effective treatment specific for CFH-associated AMD is developed, 1936 of m e dic i n e Figure 2 (facing page). Retinal Disease Associated with ABCA4 Mutations. Panel A shows a series of photographs of the retinas of patients with progressively decreasing amounts of ABCA4 function (from left to right), ranging from a normal retina to those of patients with Stargardt’s disease, cone–rod dystrophy, and retinitis pigmentosa. Panel B shows the effects of reduced ABCA4 function on fullfield electroretinograms. The relatively mild reduction in ABCA4 activity in patients with Stargardt’s disease has little effect on global photoreceptor function. Moderate loss of ABCA4 function in patients with cone–rod dystrophy has a greater effect on cone photoreceptors than it does on rods. Complete loss of ABCA4 function in some patients with retinitis pigmentosa is associated with extensive loss of both cones and rods and a nonrecordable electroretinogram. Panel C shows the effects of reduced ABCA4 function on the accumulation of bisretinoid (yellow symbols) on the inner leaflet of the photoreceptor outer segment disk membranes. Mild reduction in ABCA4 activity in Stargardt’s disease is associated with some bisretinoid formation; moderate loss of function in cone–rod dystrophy is associated with intermediate amounts of accumulation; and complete loss of function in retinitis pigmentosa results in maximal accumulation. Panel D shows the histopathological effects of reduced ABCA4 activity. In patients with Stargardt’s disease, the rate of bisretinoid formation in the outer segments is relatively slow and the photoreceptors are not directly injured. Bisretinoids are delivered to the secondary lysosomes of the retinal pigment epithelium (RPE) during the normal phagocytosis of photoreceptor outer segments. Some of this material accumulates beneath the RPE as accumulations known as pisciform flecks that are visible on ophthalmoscopy. In patients with cone–rod dystrophy, moderate loss of ABCA4 function results in sufficient accumulation of ­bisretinoids in photoreceptor outer segments to cause some apoptosis of photoreceptors (in cones more than rods). In patients with retinitis pigmentosa, complete loss of ABCA4 function causes extensive accumulation of bisretinoids in photoreceptor outer segments, apoptosis of both rod and cone photoreceptors, and associated RPE thinning. there will be little clinical benefit in a genetic test that is less sensitive and specific for the detection of AMD than a routine eye exam. Glaucoma is the second leading cause of blindness in the United States and the leading cause of blindness among blacks. As many as 60 million people worldwide currently have glaucoma.32 The most common form of glaucoma in the United States is primary open-angle glaucoma, which is characterized by optic-nerve damage and loss of peripheral visual field. Studies of mendelian (monogenic) forms of the disorder have implicated two genes (MYOC and OPTN) and mapped the chromosomal location of an additional 13 genes.33 n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. genomic medicine Normal A Stargardt’s Disease Cone–Rod Dystrophy Retinitis Pigmentosa B Rods Cones C ABCA4 Outer segment disk Partially functional ABCA4 ABCA4 absent Bisretinoid Plasma membrane D Rods Cones RPE Bisretinoid Pisciform fleck However, less than 5% of cases of primary open- proportion of the remaining cases are caused by Draft 13 angle glaucoma have been attributed to mutations the combined actions of multiple variant genes Author Stone in these single genes, which suggests that a large and environmental influences. Each variant gene 2 Fig # COLOR FIGURE 5/02/11 Title n engl j med 364;20 nejm.org may 19, 2011 ME DE Artist Phimister Knoper 1937 The New England Journal of Medicine AUTHOR PLEASE NOTE: Figure has been redrawn and type has been reset Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Please check carefully Copyright © 2011 Massachusetts Medical Society. All rights reserved. Issue date 5/19/11 The n e w e ng l a n d j o u r na l A Microtubule Ciliary membrane IFT particle Kinesin 2 Dynein Transitional fiber Basal body BBS3 B BBSome BBS6/10/12 and CCT complex BBS4 BBS8 BBS2 BBS7 BBS9 m e dic i n e Figure 3. The Bardet–Biedl Syndrome. Panel A shows the role of seven Bardet–Biedl syndrome (BBS) proteins (BBS1, 2, 4, 5, 7, 8, and 9) that along with BBIP10 form a protein complex known as the BBSome. The BBSome plays a role in cargo transport to and out of the cilia and potentially to other membrane compartments. BBS3, which is not part of the BBSome, is required for BBSome transport to cilia. Panel B shows the known physical interactions of the components of the BBSome protein complex, as determined by coimmunoprecipitation experiments. The formation of the BBSome requires a second complex, which includes proteins BBS6, 10, and 12. CCT denotes chaperonincontaining T-complex polypeptide, and IFT intraflagellar transport. Cilium Cargo of BBS1 BBS5 probably contributes a relatively small risk of primary open-angle glaucoma on its own but in unfavorable combinations may tip the balance toward the development of disease. Recently, the first such risk factor was mapped in a genomewide association study to a region of chromosome 7q31 that spans the genes encoding caveolin 1 and caveolin 2. The causal mutation in this region, which has not yet been discovered, confers a population attributable risk of at most 12%.34 This relatively small effect size suggests that the genetic basis of primary open-angle glaucoma involves the contribution of more genes with smaller population attributable risks than have been found in studies of AMD. Fuchs’ corneal dystrophy is an age-related corneal disease that affects about 5% of the population over the age of 40 years and is the leading cause of corneal transplantation. This disorder is characterized by a gradual loss of cells from the endothelial surface of the cornea, the development of small excrescences known as guttae in the underlying basement membrane, and eventual thickening and clouding of the corneal stroma. Recently, a genomewide association study showed that alleles of the transcription factor 4 gene (TCF4), which encodes a member of the E-protein family (E2-2), are strongly associated with typical age-related Fuchs’ corneal dystrophy.6 The likelihood of the disease among persons who are homozygous for the risk allele is 30 times the likelihood among persons who do not have the risk allele. However, unlike the role of CFH in AMD, in which the most strongly associated single-nucleotide polymorphism (SNP) actually alters the CFH protein in a functionally meaningful way, there is COLOR FIGURE 5/02/11 Draft 4 1938 Author Fig # Stone 3n engl j med 364;20 nejm.org may 19, 2011 Title The New England Journal of Medicine Downloaded from nejm.orgME on October 1, 2016. For personal use only. No other uses without permission. DE Phimister CopyrightArtist © 2011 Massachusetts Medical Society. All rights reserved. Knoper AUTHOR PLEASE NOTE: genomic medicine currently little corroborating biologic evidence to support the involvement of TCF4 in Fuchs’ corneal dystrophy. For example, the linked SNP lies within an intron of TCF4 and is unlikely to alter TCF4 expression. Also, persons with de novo loss-offunction mutations in TCF4 have a severe neurologic disease that does not have corneal endothelial dysfunction as a feature.8,35 Thus, as with many findings obtained through a genomewide association study, more work is needed to unravel the mechanism through which the statistically associated locus is linked to the corneal phenotype. Ther a pie s for Gene t ic E y e Dise a se A B Physicians have sought to treat inherited eye diseases at every level of the disease process — ranging from a very specific inhibition of a single mutant allele with a small inhibitory RNA molecule36 to a broad alteration of the metabolic milieu with the use of a cocktail of vitamins with an uncertain mechanism of action.37 Gene discov200 µm ery experiments have aided in this effort by providing an improved understanding of specific C biologic pathways that when perturbed lead to disease or susceptibility to disease. Such pathways can become important targets for therapeutic agents, and scientists have been very creative in devising treatments aimed at those targets. For example, the discovery that ABCA4 is involved in transporting vitamin A derivatives out of outer segment disks16 led to the discovery that 200 µm vitamin A inhibitors such as fenretinide inhibit the accumulation of lipofuscin in animal models Figure 4. Treatment of Age-Related Macular Degeneration (AMD) of Stargardt’s disease.38 The identification of the with Bevacizumab. role of vascular endothelial growth factor in choA retinal photograph of a 67-year-old patient with a subretinal neovascular membrane (Panel A) shows yellow deposits (drusen) beneath the retinal roidal neovascularization led to the development pigment epithelium (arrows), which are the clinical hallmarks of AMD. of therapeutic antibodies (e.g., ranibizumab and Cloudy subretinal fluid and small hemorrhages in the center of the macula bevacizumab) to combat the major blinding comare suggestive of subretinal neovascularization. The horizontal black line plication of AMD (Fig. 4). (An article describing indicates the center of the macula. The visual acuity in this eye is 20/80. a test of noninferiority of these drugs in the A spectral-domain optical coherence tomogram (SDOCT) taken through the center of the macula reveals subretinal neovascular tissue and fluidtreatment of AMD appears in this issue of the filled spaces within the retina (Panel B). A repeat SDOCT taken after three Journal.39) Growth factors40 and neuroprotective intravitreal injections of bevacizumab during a 3-month period reveals a 41 agents have also been used to reduce the rate dramatic reduction of neovascular tissue and intraretinal fluid (Panel C). of an apoptotic response to inherited cellular The visual acuity has improved to 20/50. abnormalities. In recent years, gene-replacement therapy, therapeutic stem cells, and retinal prostheses have also moved to the threshold of clini- es is that a complete knowledge of the specific cal use for the treatment of genetic eye disease. molecular cause of a patient’s disease may not be A potential advantage of the latter two approach- necessary for the treatment to be successful. n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. 1939 The n e w e ng l a n d j o u r na l Gene Ther a py Most human photoreceptor diseases are inherited in an autosomal recessive manner, and in these cases the mechanism of disease usually involves a profound loss of function of the gene product. More than a dozen recessive retinal diseases have been successfully treated with viral- or nanoparticle-based gene transfer in animal models.42 For example, one molecular form of Leber’s congenital amaurosis is caused by the lack of the retinoid isomerase encoded by RPE65. A decade ago, Acland and colleagues43 successfully restored vision in a naturally occurring canine model of this disease using an adeno-associated viral vector to transfer a normal version of RPE65 to the retinal pigment epithelium. More recently, three different groups have extended this work to humans.44-46 For example, 3 years ago, Maguire and colleagues44 reported results in 12 patients who were treated with gene-replacement therapy. They observed visual improvement in all 12 patients, with the greatest gains among younger patients. T r a nspl a n tat ion of S tem Cel l s Several important cell types in the eye have little if any capacity for endogenous regeneration, and as a result the only viable treatment option for patients with hereditary disorders that involve the loss of such cells is some type of cell-replacement therapy. Although the replacement of highly differentiated cells, such as photoreceptors, poses challenges, a number of recent experiments suggest that the use of stem cells to achieve such a goal is now feasible. In 2004, Klassen and colleagues47 found that transplanted retinal progenitor cells could develop into functional photoreceptors and give rise to enhanced visual function in mice with retinal degeneration. Since these original reports, an assortment of different cell types, ranging from the fate-restricted photoreceptor precursor to the pluripotent embryonic stem cell,48-51 have been used to replace photoreceptors in animals with inherited retinal disease. Embryonic stem cells have been of particular interest because of their ability to undergo unlimited self-renewal and tissue-specific cell production. For instance, Eiraku and colleagues52 recently found that by using a three-dimensional cell-culture system, they could recapitulate development and reliably gen1940 of m e dic i n e erate functional photoreceptor cells in vitro. These properties permit the generation of cells in sufficient numbers to perform clinical transplantation from a single isolation rather than the multiple donations that are required when more developmentally restricted cell types are used. Regardless of the theoretical utility of these cell types in humans, the isolation of cells from human embryos is rife with ethical concerns and immunologic limitations. As a result, freshly isolated embryonic stem cells seem unlikely to be widely used in the treatment of degenerative eye disease. A cell type that overcomes the majority of these limitations is the induced pluripotent stem cell (iPSC). Initially produced by Takahashi and Yamanaka 5 years ago,53 iPSCs can be generated by genetic re­programming of dermal fibroblasts to pluripotency through retroviral transduction of only four transcription factors.53 Several groups of investigators have been able to show that iPSCs have the capacity to generate a variety of retinal-cell types, including photoreceptors,54,55 and that after transplantation these photoreceptors will integrate with­in the dystrophic retinal architecture,56,57 which results in partial recovery of the electroretinographic response.57 Although methodologic barriers, such as the use of retroviruses, preclude the immediate clinical translation of this technology, and recent studies suggest that the process of somatic-cell reprogramming may result in the introduction of pathway-specific genetic defects,58-60 this field is evolving rapidly, and it is possible that these cells will eventually make their way into clinical use. R e t ina l Pros the se s In normal vision, decreased glutamate at the photoreceptor axon terminals stimulates bipolar and amacrine cells, which in turn release glutamate to stimulate the ganglion cells that communicate with the brain. In an attempt to bypass photoreceptors and other neuronal elements that have been damaged by degenerative retinal disease, investigators have explored the possibility of stimulating the ganglion cells directly with the use of electrical impulses delivered from a planar array of microelectrodes. Several different designs for retinal prostheses have had promising results in both animals and humans,61 and one of these designs has recently been approved for clinical use in Europe. n engl j med 364;20 nejm.org may 19, 2011 The New England Journal of Medicine Downloaded from nejm.org on October 1, 2016. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. genomic medicine C onclusion The eye has figured prominently in the development of genetic and genomic approaches to human disease. Vision is critically important to most activities of daily living, and cures for blindness will remain an important goal for medicine for many years to come. Physicians and scientists will be aided in the pursuit of this goal by the optical and anatomic accessibility of the organ, as well as by the large amount of visual cortex devoted to the interpretation of the neural information originating in the retina. That is, a patient with a disease that injures only a few thousand neurons in the fovea can describe this injury to his physician in great detail, and the physician can in turn view these neurons in the living patient at microscopic resolution by taking advantage of the near perfect optics of the anterior portion of the eye. These natural optics also contribute to a surgical accessibility that is unmatched by any other part of the central nervous system. This latter attribute will be a tremendous advantage for clinician scientists seeking to translate all the recent progress in gene-transfer and stem-cell biology into effective therapies for their patients with genetic eye diseases. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. References 1. Senju S, Haruta M, Matsunaga Y, et al. Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells. Stem Cells 2009;27:102131. 2. Azuma N, Nishina S, Yanagisawa H, Okuyama T, Yamada M. PAX6 missense mutation in isolated foveal hypoplasia. Nat Genet 1996;13:141-2. 3. Wilson EB. The sex chromosomes. Arch Mikrosk Anat Enwicklungsmech 1911;77:249-71. 4. Renwick JH, Lawler SD. 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