Age-related macular degeneration: genetics and implications for detection and treatment

Reviews MOLECULAR MEDICINE TODAY. FEBRUARY ,997

Age-related macular degeneration: genetics and implications for detection and treatment

Age-related macular degeneration (ARMD) remains the most common cause of registerable blindness in the developed world. Despite extensive research, the pathogenesis of this condition remains elusive. Recent genetic advances in the understanding of inherited retinal dystrophies and the discovery of genes that code for retinal proteins have rekindled interest in the possibility of a genetic predisposition to ARMD. ARMD is most probably a disease with a multifactorial inheritance in which environmental factors can trigger disease in those who are ‘genetically primed’. If it were possible to detect predisposing genes in these people, then perhaps novel therapies or preventive measures could be directed towards those at risk in the pre-symptomatic stage, in the hope of either preventing disease or decreasing its severity.

AGE-RELATED macular degeneration (ARMD), first described in 1885, is a progressive disabling bilateral condition which is responsible for 3049% of new blind registrations per year in the UK alone’s’. Although the condition is common, the underlying aetiology remains an enigma. Numerous potential risk factors have been causally but not definitively implicated. More recently there has been renewed interest in the possibility of a genetic predisposition in ARMD and it now appears likely that ARMD is a multifactorial disease that is triggered by environmental factors in those who are genetically predisposed. Clinically, AKMD is a heterogeneous disease and is classified into two subgroups: 80% of ARMD patients have the ‘dry’ or ‘atrophic’ form, and the remaining 20% have the exudative or ‘wet’ form3 (Table 1). Wet ARMD, although much less common, is responsible for the majority of cases of severe loss of central vision.

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MOLECULAR MEDICINE TODAY, FEBRUARY 1997 Reviews

The patient with dry macular degeneration usually gives a history of slow visual loss (Fig. la). There can be long periods when the patient’s vision is stable, interspersed with periods of slow deterioration. Although severe visual loss is possible in dry ARMD, severe loss of central vision does not usually occur until 80% of the fovea has been damaged. By contrast, patients with wet ARMD often complain of sudden onset of distorted vision, decreased near visual acuity, micropsia or scotoma (Fig. lb). The rapid onset of visual compromise can be devastat- ing to the patient both psychologically and practically. Because visual acuity often remains good in one eye, patients are often unaware that the first eye has been affected until quite late in the process, by which time any possible therapeutic window has passed. At present only 5% of patients with wet ARMD are treatable by argon laser photocoagulation.

Histopathology and pathophysiology of ARMD Histologically, dry ARMD demonstrates loss of the photoreceptors and the retinal pigment epithelium. This results in adhesion of the outer plexiform layer of the retina to Bruch’s membrane, the extracellular matrix to which the retinal pigment epithelium is normally attached. The sequential changes that occur under the macula following failure of the retinal pigment epithelium in ARMD are described in Fig. 2. The hallmark of the severe and visually destructive wet form of ARMD is choroidal neovascularization4. This neovascular response is dependent on the presence of degenerating but viable retinal pigment epithelium. New vessel complexes are therefore found close to the edge of atrophic areas. A choroidal neovascular membrane (CNVM) appears as a neovascular sprout, growing under or through the retinal pigment epithelium via breaks in Bruch’s membrane. These vessels spread along the plane created by the layers of membranous debris. As the complex matures, an orga- nized vascular system develops from a trunk feeder-vessel from the choroid. The endothelial cells of this neovascular net lack tight endothelial junctions and therefore fluid and blood leak into the neurosensory, subsensory and subpigment epithelial layers of the retina4. This event precipitates a healing, fibrotic response that causes scarring and disruption of the central vision. At present, the stimulus for this vascular ingrowth remains unknown. However, an understanding

Table 1. ClfnEcal fedures tn age-related macular degeneration

Dry Af?#ID We4 ARMD

Presenting compiaints O~~~~~~~fl Metamorphopsia Missing fetter3 in a word Sudden loss of central vision

cllfibi findings Hard drusen Hard drusen soft drusefi Soft drusen pismentary changes Serous retinal detachment Retinal ~grnent~~~~~~ atrophy Sub-retinal haemorrhage

Retinal pigment epithelial detachment Subretinal fibrosis

Figure 1. (a) Photograph of the left fundus, showing typical clinical appearance of hard drusen (white arrow), soft drusen and areas of atrophy (black arrow) in dry age-related macular degeneration (ARMD). (b) Photograph of the right fundus, showing extensive changes in advanced wet ARMD. The clinical findings include serous detachment of the retina, intraretinal and subretinal haemorrhage (white arrows) and massive intraretinal exudation (black arrow). (c) Photograph of the right fundus showing an acute extrafoveal choroidal neovascular membrane (CNVM) before laser photocoagulation (arrow). The CNVM appears as an oval, pale raised area with overlying flecks of haemorrhage. (d) The same fundus following laser photocoagulation. The laser scar is visible as a pale yellow/white area, slightly larger than the original membrane (arrow).

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Reviews MOLIXXJLAR MEDICINE TODAY. FEBRUARY 1Y97

l Hard drusen in 83% of normal adult eyes l Loss of fovea1 cells l Fall-out of cells from inner retinal layers l After 50 years: increase in lipofucsin in RPE cells

T

mineralized deposits in elastic lamina

l Hard drusen form l Hard drusen l Some hard drusen

l Pigmentary changes

breakdown with membranous debris between laminae

l Pigment clumps l Areas of atrophy form l Atrophy centred on fovea

l RPE atrophy l Focal loss of retinal receptor

cells and RPE l Choroidal neovascularization l Pigment epithelial detachment l Disciform scar

Figure 2. The sequential changes found under the macula during evolution of age-related macular degeneration (ARMD). WE, retinal pigment epithelium.

of the underlying mechanisms is crucial to the development of an effective treatment strategy in wet ARMD.

Some of the most exciting recent developments in ARMD have been in the field of pathophysiology. Recent work describes some interesting correlations between the chemical composition of drusen (the dense, rounded, homogeneous, hyaline bodies beneath the basement membrane of the retinal pigment epithelium) and clinical features in macular degeneration. Although the nature of the stimulus for choroidal neovascularization remains unknown, there is now evidence that the chemical composition of the deposits in Bruch’s membrane, the metabolic environment of the retinal pigment epithelium and the conductivity across Bruch’s membrane might all be important in causing detachment and subsequent atrophy of the retinal pigment epithelium5x6. The retinal pigment epithelium is responsible for pumping fluid from the retina towards the choroid, and recently Bird and co-workers have shown that the deposition of hydrophobic lipids in Bruch’s membrane results in a decrease in the permeability of the membrane which, in turn, can reduce the efficiency of the pumping process, leading to detachment of the pigment epithelium6. Detachment of the retinal pigment epithelium is an important feature of ARMD in some patients, and it might interfere with the metabolic function of both the choroid and pigment epithelium, resulting in atrophic changes. However, the stimulus for subsequent choroidal neovascularization remains unknown. If the stimulus for this destructive process were known, the development of preventive therapy would be a more realistic option.

Risk factors for ARMD Although multiple possible environmental risk factors for ARMD have been identified, the importance of each remains uncertain’. Those thought to be important include: demographic characteristics;

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personal history of certain medical diseases; ocular status; personal attributes such as eye colour and race; and environmental factors such as light exposure, cigarette smoking, alcohol consumption, and, more recently, ingestion of plasticizersc9 (Table 2). Nutritional factors such as intake of vitamins A, C and E have been shown to have some possible protective function in ARMD owing to their antioxidant properties”. This has led to the hope of using these vitamins either for prevention of disease or to halt the progression of established disease.

Treatment strategies The treatment of age-related macular degeneration remains a difficult problem for both the patient and the physician. Despite the high prevalence of ARMD, the ability to influence this disorder by either prevention or cure remains depressingly poor. The various manage- ment options include: medical therapies such as interferon a2a; laser photocoagulation of the leaking neovascular membrane; and, more recently, surgical excision (or irradiation) of these lesions. Although laser photocoagulation has been the mainstay of treatment over the past 15 years, only 5% of patients presenting with wet ARMD are suitable for therapy, and 60% of those treated experience recurrence of their pathology within three years”. The reason why only such a small number of patients are eligible for laser treatment is that laser treatment has only been shown to be of real benefit when the neovascular membrane lies a certain distance from the fovea (Fig. 3a). At present, owing to the poorly understood pathogenesis of dry ARMD, there are no treatment options for this form of the disease. The interest in unravelling the genetics of this condition lies in the hope that, in the future, genetic screening of those at risk combined with possible dietary, environmental or therapeutic manipulation will offer new opportunities for treatment.

Genetic iniluence in ARMD Recently, an increased interest in the genetics of ocular disease and a dramatic improvement in molecular-genetic techniques have resulted in a renewal of interest in the possible genetic predisposition in common diseases of adult life, such as ARMD. The first documentation of a genetic input into ARMD dates back to 1875, when Hutchinson and Tay described three patients with familial drusen’*. It is conceivable that this is the earliest report of ARMD showing a familial tendency. In 1966, Bradley also reported a positive family history of ARMD in his patients, as did Gass in 1973 (Refs 13, 14). In 1981, Hyman reported a 2.9-fold increased risk of ARMD if one or more family members had the condition (L.G Hyman, PhD Thesis, The Johns Hopkins University, I98 1). Although these studies are indicative of a genetic influence in ARMD, until recently little work had been carried out to investigate further the implication of hereditary factors in this disease. This might be related in part to the perceived (and very real) practical difficulty in studying ARMD on a genetic level.

Although many hereditary diseases demonstrate a simple mode of inheritance, some diseases show familial traits that are so complex that the mode of inheritance is difficult to decipher. ARMD and other common chronic diseases such as diabetes, hypertension, arthritis, coronary artery disease and schizophrenia present both a great bur- den to human health and resources, and a great challenge to the molecular geneticist. These diseases are thought to be characterized by a complex multifactorial aetiology, indicating that a number of disease- predisposing genes combine with many different environmental trig- gers to determine an individual’s level of resistance or susceptibility to developing disease. The study of the genetics of ARMD presents a particular problem owing to the lateness of disease expression. As ARMD typically presents when an individual is in his/her late 60s or 7Os, clinical examination of the parents of the affected patients is virtually impossible. Detection of ARMD in children of affected parents is also very dil%cult until they reach the typical age of expression of the disease. As a result, the study of genetic implications in ARMD requires a different approach from that used with diseases that demonstrate Mendelian characteristics. The methods available for studying ARMD are discussed below.

Statistical methods for studying the genetic influence in genetically complex diseases The methods used for the molecular study of diseases that are inherited in a Mendelian manner are now well known. There are two ways of dealing with a disease process that as yet has not been genetically approached: the ‘linkage’ method and the ‘candidate gene’ method. Linkage can be carried out only when a good family pedigree is available, whereas the candidate gene approach can be used when the family pedigree is poor but a suitable (retinal) candidate gene is available for study. To date, linkage studies have been very successfully applied to diseases that have a well-defined mode of inheritance. Methods for statistical analysis that extend the linkage method to map the underlying

genes responsible for traits with complex inheritance have also been developed, but their accuracy remains to be verified”,“. Currently used approaches to genetically complex diseases include the affected- relative pair method and the calculation of the heritability score of a disease17-19 . A third approach, the sib-pair method of linkage analysis, which is based on the affected-relative pair method, provides a feasi- ble approach to a complex, late-onset disease such as ARMD’*. It is estimated that at least 200 affected sib-pairs would be required for a statistically significant calculation of linkage to a genetic locus using the affected-relative pair method.

Application of the concept of heritability to ARMD The evidence that genetic factors are important in common diseases comes mainly from epidemiological studies that compare the fre- quency of disease among genetically related individuals with that in the general population. The concept of heritability was developed to allow a quantitative measure of innate genetic predisposition to a disease. By definition, heritability is the proportion of the total pheno- typic variance of a trait that is caused by additive genetic variance19; variance is a measure of how much an individual value is likely to vary from the mean of a group. Heritability is a useful concept because even when the actual genetic basis of a disease such as ARMD is not known, the importance of genetic factors in its causation is indicated by the heritability score of the disease*“. The higher the heritability score, the more important the genetic input in the disease”. If the disease is determined chiefly by the environment, the value of the heritability score approaches zero. Recently, a heritability score for ARMD has been calculated by comparing the incidence of disease in the siblings of affected patients and age-sex-matched control subjects*‘.

Fifty patients diagnosed consecutively with either wet or dry ARMD were identified using fluorescein angiography records (to avoid bias in selecting patients who were known to have a positive family history of the disease). Both dry and wet macular degeneration cases were

Table 2. Studies investigating risk factors for age-related macular degeneration (ARMD)

Reference

Risk factors 1 7 8 9 38 39 b

Hypennetropia * NA t ’ t t +

Decreased hand-grip strength ’ l l t * t

+

Iris colour * NA l * * * +

Elevated blood pressure t NA t + + - -

Positive family history of ARMD l + * l - * t

Decreased vital capacity l * t +

t l l

Left ventricular hypertrophy * * * + t l t

Short stature * * NA + l * -

History of lung infection t NA l + ( I

Cigarette smoking * NA l - * - +

History of cardiovascular disease * NA t * l l +

Chemical exposure at work l l * ’ * l +

Dietary factors + NA - * * * -

aKey: ‘, Not studied; c, positive association found; -, negative association; NA, no significant association found.

bL.G. Hyman, PhD Thesis, The Johns Hopkins University, 1981.

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Reviews MOLECULAK MEDICINE TODAY. FEBRUARY ,997

a Juxtafoveal CNVM Subfoveal CNVM

yq\\ Perifoveal

:one

capillary network r Extrafoveal CNVM

Figure 3. (a) Schematic showing the perifoveal capillary network and the fovea1 avascular zone. Three types of choroidal neovascular membranes (CNVMs) are depicted: an extrafoveal CNVM (yellow), which lies >200 pm from the fovea; a juxtafoveal CNVM (orange), which lies ~1 but 499 pm from the fovea; and a sub- fovea1 CNVM (red) that lies directly under the fovea. Laser photocoagulation has only been proven to be efficacious in extrafoveal CNVM. (b) Photograph of a normal fluorescein angiogram, showing the right fundus and a normal macular area. (c) Photograph of a fluorescein angiogram showing a leaking subfoveal CNVM in a 70-year-old patient who has noticed acute visual distortion. This lesion is analogous to the subfoveal lesion (red) in (a) and would be unsuitable for laser photocoagulation because of its proximity to the centre of the fovea.

included and ‘single incomplete ascertainment’ was used. The criterion for inclusion was that the patients should have a visual acuity of 6/9 or less attributable to ARMD-related changes. The heritability score from this study was found to be 118% + 19% (the range was 99-137). This high score suggests that the degree of genetic determi- nation in ARMD is of prime importance and that environmental factors have a secondary triggering influence. However, the estimation of heritability is only meaningful if there is no genetic heterogeneity in the disorder being studied, and if no major gene is contributing to the causation of the disease. If a major gene contributes to the causation of the disorder, the method will break down because the continuous variation of liability no longer holds and the estimated heritability can therefore exceed 100%. This is possible in this study. In addition, members of the same family are likely to be exposed to the same environmental factors and their liability to disease can therefore correlate for purely environmental reasons. This source of error tends to affect siblings more than other relatives but must be

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recognized if the score is very high. Therefore, accurate estimates of heritability will not be based on data from siblings alone. The heritability score in this study is based on full siblings alone and, as such, could cause a falsely high result.

Over the past three years, several studies have provided further support for the genetic influence in ARMD. In a study comparing characteristics of drusen (early clinical markers of ARMD) in the spouses and siblings of affected ARMD patients, Piguet and co- workers revealed marked concordance between siblings but not between spouses, suggesting that genetic factors are more important than the environmental factors shared by spouse?‘. Two recent studies have assessed the concordance of fundal changes for ARMD in monozygotic and dizygotic twins23,24. Both studies show a high rate of concordance for fundal findings in ARMD in the monozygotic pairs (23/23 pairs in one study and 8/9 pairs in the other). The level of concordance was much less striking in the dizygotic pairs, as would be expected. Sibling correlations for fundal changes in ARMD

MOLECULAR MEDICINE TODAY, FTBRUARY 1997 Reviews

Table 3. Retinal candidate genes

Candidate gene LOfXS

S-antigen Rhodopsin u Rod transducin a Subunit of cGMP phosphodiierase 6 Subunit of cGMP phosphodiisterase Peripherin-RDS lntraretinai binding protein Retinal binding protein Rod outer-membrane protein 1 Recoverin Retinal guanylate cyciase Arrestin Pigment epithelial-derived growth factor Tissue inhibitor of metailoproteinases 3

z 3s 3s 4p16.3 6p21.1-CX3n

lop 10s llq13

l7P t7P 17P t7P 22ql3qter

have also been performed in the 564 families in the Beaver Dam Eye Study. This study also showed significant sibling correlation for fun- da1 findingsz5. A case-controlled study has also investigated the role of a positive family history of ARMD in siblings; this revealed that the risk of developing ARMD was 19 times greater for the sibling of an affected person than for the sibling of an unaffected contro12’. Examination of the affected pedigrees in the families used in the latter study revealed a positive familial trait (defined as two or more family members with ARMD) for ARMD in 58.3% of families with an affected proband2’.

The role of molecular genetics in inherited retinal diseases The first significant advance in the understanding of those human retinal diseases that are inherited in a Mendelian fashion began in 1989 when Humphries and co-workers mapped the first locus for autosomal-dominant retinitis pigmentosa to chromosome 3q in an Irish kindred26. This was quickly followed by the identification of mutations within the protein rhodopsin*‘. Rhodopsin is located in the rod photoreceptors, is a primary component in the visual cycle and is responsible for night vision (Fig. 4). To date, in the dominant form of retinitis pigmentosa at least 80 mutations have been identified in the gene encoding rhodopsin’s. Retinitis pigmentosa, now known to be a very heterogeneous condition, has since been linked to loci on several different chromosomes29+30. Since 1989, many other inherited retinal and vitreoretinal disorders have been mapped to various site?‘. To date, the most commonly implicated retinal proteins in retinal diseases are peripherin-RDS and rhodopsin. However, this might simply reflect the fact that these two genes have been available for study for some tiieW (Fig. 4). Rhodopsin has been implicated mainly in the causation of peripheral retinal dystrophies, whereas peripherin-RDS has been implicated in causing both peripheral and central dystro- phies3’. As the gene for peripherin-RDS is expressed in both rods and cones, it is a much more likely candidate gene for maculopathies and has been implicated as the diseasecausing gene in a number of disease$‘. The macular dystrophies that have so far been mapped all share some histological and clinical features with the more common ARMD. Research in the field of molecular genetics on juvenile macular dystrophies and retinitis pigmentosa over the past five years has been

prolific, with the result that the list of known retinal candidate genes and retinal disease loci continues to increase steadily (Table 3). This has provided a stimulus for further molecular investigation into the more common but genetically less well-defined ARMD. It is hoped that the elucidation of the precise pathogenic mechanism for these juvenile macular dystrophies will help shed some light on the pathogenesis of the age-related type.

Genetic models for ARMD One of the most promising recent discoveries that might help in the study of ARMD is the mapping of the rare autosomal-dominant Sorsby’s fundus dystrophy (SFD), which shares many clinical and pathological features with ARMD3’. Recently, SFD was mapped to the locus 22q13qter (Ref. 32). This region was also known to contain the gene for tissue inhibitor of metalloproteinases 3 (TIME-3), which plays a central role in extracellular-matrix modelling, and mutations in TIMI- have now been identified in SFd3. Both of the mutations found in the TZMP-3 gene in SFD cause a substitution of amino acids, which would be expected to influence the structure and perhaps the properties of TIME-3. Interestingly, both SFD and ARMD show pathological changes in Bruch’s membrane. It is possible, therefore, that the integrity of Bruch’s membrane could be influenced by the compromised action of a mutated TIhW3, perhaps leading to the onset of the disease state of SFD and ARMD3*. Recently, however,

Glossary

Bruch’s membrane - An extracellular matrix lying between the retinal pigment epithelium and the choroid.

Choroidal neovascular membrane -A neovascular sprout, growing under or through the retinal pigment epithelium via breaks in Bruch’s membrane.

Drusen - Dense, rounded, homogeneous, hyaline bodies beneath the basement membrane of the retinal pigment epithelium. Hard drusen are found in the normal ageirtg eye but can also be the first sign of AND. Soft drusen indicate progression of ARMD.

Fluorescein angiography - A dye test used to show the circu- lation of the retina and any diseases present.

Metamorphopsia - Distortion of an image in the central visual field.

Micropsia -A visual image that is diminished in size.

Scotoma -An area of the visual field that is blanked out.

Serous detachment - Detachment of the neurosensory retina by fluid and/or blood.

Single complete ascertainment - A method of sampling for genetic studies so that every family sampled has one proband.

Sub-retinal fluid - Fluid gathering beneath the neurosensory retina, causing a serous detachment.

Visual acuity - The ability of the eye to resolve detail. This is measured by a standard ratio or fraction comparing patient perfor- mance against an agreed-upon standard; 616 is nomal visual acuity by the Snellen notation.

Reviews MOLECULAR MEDICINE TODAY. FEBRUARY ,997

-rPDE /,/

l cx,f3 PDE

H+ + 5’-GMP J

Rod outer segment u I I I I

Figure 4. Schematic depicting (in cross section) a rod photoreceptor outer-segment and adjacent retinal pigment-epithelial (RPE) cell. Outer-segment proteins that are associated with or suspected of involvement in human retinopathies are shown. Abbreviations: PDE, cGMP phosphodiesterase; TD, transducin (a, p and y denote the three subunits of each of these proteins); ROM-l, rod outer-membrane protein 1. Modified from Ref. 30.

Seddon et al. reported a linkage/sib-pair study showing no association between ARMD and the TMP-3 gene3”.

It would be interesting to know whether patients with ARMD demonstrate any disease-causing mutations in any of the other known retinal candidate genes. One study has examined peripherin-RDS for disease-causing mutations in 50 patients affected by ARMD and in 50 age-sex matched control subjects, using mutation detection analysis. No disease-causing mutations in peripherin-RDS were found in either the affected patients or the controls. Polymorphisms in the highly polymorphic third exon of the protein were found to be equally common in the affected patients and the controls (G. Silvestri, MD Thesis, The Queen’s University of Belfast, 1994).

Difficulties in the genetic study of ARMD The very late expression of disease in ARMD provides the most substantial hindrance in studying this disease, because large, multigeneration families are impossible to find. ARMD might well have a truly genetic basis, in that risk is primarily determined by disease-

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causing genes, but other factors might be required either to trigger the disease process or to modify the age of onset, disease type and severity of expression. Another possible approach to the molecular investigation of ARMD is to study the fairly common condition of dominantly inherited druser?. In this condition, the drusen become clinically manifest when the subject is in his/her 2Os, allowing the geneticist to construct multigeneration pedigrees that would facilitate linkage studies. As drusen are the hallmark of ARMD, perhaps elucidation of the genetics of dominantly inherited drusen would contribute to the understanding of the genetics of the most common forms of ARMD. It might also lead to the identification of the molecular mechanisms of druse formation.

Future prospects for ARMD The prevalence of ARMD has shown a steady increase over the past few years, and recent analysis of those registered as blind has shown that this is not simply due to the increasing age of the population36. Although at present the future for those already afflicted by ARMD holds little promise for treatment, it is important that that research to under- stand the pathogenesis of this condition continues. ARMD, like many retinal diseases, appears to be an extremely heterogeneous condition with a large spectrum of clinical presentation. It is now evident that one factor alone is unlikely to be responsible for ARMD and also that genetic predisposition is likely to be modified by environmental factors. Because ARMD is very heterogeneous, it is conceivable that some forms could be caused by single gene defects with strong environmental influences, and other forms might be polygenic without environmental input. It is

interesting that one form of autosomal-dominant retinitis pigmentosa has been found to be digenic (two genes must be inherited before there is expression of the disease), and it is conceivable that a similar, but perhaps more complex, polygenic situation could apply to some forms of ARMD37.

One possible treatment strategy for the future would be the identification of genetic markers that could identify those at risk of ARMD at the pre-clinical stage. If it were possible to identify those at risk, it might then be possible to modify other (as yet unknown) risk factors, or perhaps intervene in the disease process by modifying the expression of disease-producing genes. At this very preliminary stage, the possibility of gene therapy for patients with ARMD seems virtually impossible. However, there has been rapid progress in the understanding of retinitis pigmentosa and other retinal dystrophies at the molecular level, and it might be possible to use gene therapy for some of these diseases. This should provide us with the technical tools to deliver such therapy for ARMD. The delivery methods for gene therapy of retinal degeneration are most likely to be local; however, systemic therapy

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Age-related macular degeneration: genetics and implications for detection and treatment