Age-related macular degeneration (AMD) is a common, progressive and potentially blinding retinal disease, which mainly affects the elderly population. It has been estimated that 30% of the individuals over 75 years of age manifest this condition and 6-8% of them suffer its advanced forms involving severe central loss of vision 1. AMD is the first cause of blindness worldwide in the elderly population 7.
With age, the ocular tissues experience typical modifications such as changes in size and shape of retinal pigment epithelium (RPE) cells, thickening of the Bruch's membrane as well as of the internal limiting membrane, and accumulation of subretinal deposits made of glycoproteins and lipids 2. The latter along with RPE changes can result in the formation of drusen. While their presence in small number in the peripheral retina generally occurs as consequence of the physiological ageing process, the localisation of many drusen within the macula is likely to indicate AMD 2 (figure 1 and 4a). Drusen are divided into the nodular, well-defined hard drusen and the less definite soft drusen. The latter are more frequently associated with severe AMD cases such as geographic atrophy (GA) and choroid neovascularisation (CNV) 4. In CNV (figure 2 and 4b), new vessels grow from the choriocapillaris through the Bruch's membrane under the RPE, often leading to profound visual loss following haemorrhages, oedema and/or retinal detachment, as a consequence of mechanic forces. This form is called wet (exudative) as opposed to dry (non-exudative) AMD which involves no formation of new vessels. The end stage of dry AMD is GA (figure 3), characterised by RPE hypo- and hypertrophy, degeneration of the Bruch's membrane and of the choriocapillaris, etc. The deterioration of the above described tissues in both CNV and GA results in impaired function first, and subsequently death of the macular photoreceptors, often causing severe loss of the central vision.
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The concentration of inflammatory molecules within drusen 5 as well as the presence of leukocytes and activated complement factors 6 in the retinal tissues of AMD mouse models, suggest a key contribution of inflammatory components in the pathogenesis of the disease.
The familiarity showed in previous studies 7, 8 suggested an important genetic influence in the development of AMD. This has been confirmed in recent years through the discovery of multiple genes which, in different proportion, play a role in the various forms of this condition. In addition, environmental factors such as smoking, alcohol abuse, sunlight and some microbial infections have been associated with the disease while lutein and zeaxanthin as well as omega-3 fatty acids have been shown to have a protective effect towards the development of AMD 3.
In summary, age-related macular degeneration is a complex disease, and its pathogenesis is influenced by multiple genes variants, the integration of their products with many further molecular elements as well as different environmental factors. The sequencing of the entire human genome and the recent technological advances in genotyping, allow a new insight into the nature of this complex disease, increasing our discrimination ability between the different polygenetic and environmental contributing factors.
Figure 1a: photograph of the left fundus showing soft drusen suggesting intermediate AMD 3.
Figure 1b: optical coherence tomography (OCT) of the above eye. Drusen cause a "wavy" RPE pattern 3.
Figure 2a: photograph of the right fundus of a patient affected by severe AMD with CNV. Subretinal haemorrhages, hard exudates and large drusen are present 3.
Figure 2b: fluorescein angiogram of a patient with neovascular AMD (exudative from) 3.
Figure 3a: fundus photograph showing GA, a severe form of non-exudative AMD 3.
Figure 3b: OCT of the above patient showing loss of RPE and choriocapillaris in the central macular region 3.
Figure 4a: schematic representation of an early form of AMD. The arrow shows a large drusen 3.
Figure 4b: schematic representation of a severe form of AMD. The arrow indicates the new choroidal vessels growing through the RPE. The photoreceptors are severely damaged 3.
Techniques of genetic analysis
Linkage analysis has been more successful in detecting the genetic basis of diseases caused by a single gene mutation rather than of complex disorders such as AMD. Nevertheless, this technique allowed the determination of the central role of genetic variants localised closed to CFH and ARMS2/HTRA1 in the pathogenesis of AMD 3. Klein et al, for example, discovered the connection between a form of dry AMD and chromosome 1q25-31 10. In addition, investigation of several genomes found AMD related loci on chromosomes 1q31 and 10q26 11.
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In recent years more effective methods of analysis have been developed to identify the origin of complex diseases, which dissect the genetic from the environmental influencing factors. For example, single nucleotide polymorphisms (SNPs) across the genome of AMD subjects can be compared with datasets made of the DNA pool of non-affected individuals 12, enabling the detection of genetic variants involved in the pathogenesis of the disease. This method, known as genome-wide association study (GWAS) 14, is now available thanks to the most recent technological advances in DNA sequencing and mapping techniques.
This process has been successfully applied to other ocular disorders such as glaucoma13 as well as to common medical conditions like cardio-vascular pathologies, many forms of cancer, diabetes mellitus, etc.
Factors involved in the complement cascade
Several SNPs have been discovered to lie in genes responsible for inflammatory factors related to the complement cascade; moreover many studies demonstrated a relationship between these elements and the pathogenesis of AMD 15.
Complement factor H (CFH) is a member of the complement cascade. The presence of CFH polymorphism is linked to AMD in more than 50% of the affected subjects 2. The encoding gene is located on chromosome 1q23-32 and its locus was related to AMD in one of the first linkage studies performed on this disease 10. An important variant of CFH is Y402H (tyrosine to histidine substitution at amino acid 420). CFH is found in normal ocular tissues such as the choroid and the RPE 16. Its function is to limit the alternative pathway by supporting the inhibition of C3b as well as by splitting Bb from the C3b-Bb complex 17. A debate is still open on the association of CFH polymorphism and CRP. Some studies support this theory based on the fact that homozygous subjects for Y402H CFH have elevated CRP concentrations in the choroid 18. Other publications, in contrast, showed how CFH primarily connects to the denaturated instead of the primitive CRP 19.
In addition, AMD was also associated with Chlamydia pneumoniae infection 20. Some studies also demonstrated an additive interaction between AMD-related CFH variants and this infective disease, resulting in an increased combined risk which is higher than the single contributions of the Chlamydia pneumoniae infection and the CFH genotype respectively 21.
Cfhâˆ’/âˆ’ mice in contrast to its control animal showed impaired visual acuity, reduced electroretinogram, disruption of the photoreceptors outer layer and high C3 concentration. The latter is normal in the AMD genetic variants affecting human subjects 26.
Further genotypic variants encoding for many other factors involved in the complement cascade such as complement factor B (BF), complement component 2 (C2), C3, C7, and complement factor I (FI), have been shown to be involved in the pathogenesis of AMD. Similarly to CFH, while defect complement inhibition leads to an increased risk, impaired activation results in AMD protection 2. For example, after a relationship between the adjacently located C2 and BF on chromosome 6p21.3 and AMD had established through linkage studies, SNPs analysis identified a degree of protection given by L9H BF/E318D C2 and R32Q BF/intronic variants of C2 against the disease 22. On the other hand, variations of the C3 locus on chromosome 19p13 as well as near the FI gene on chromosome 4q25 have been associated with an enhanced risk of developing AMD 23, 24. The biological function of FI is to degrade the C3b component of the C3bBb complex. Finally, it was discovered that C7 (rs2876849) has also a protective function 25.
Chemokines and their receptors
Chemokines, after binding on receptors of inflammatory cells, attract them towards inflamed tissues.
CX3CR1 is a G-coupled receptor which has been shown to have an important role in the pathogenesis of AMD. It is placed within the retina and, after attachment of its corresponding ligand CX3CL1, promotes the activation and mobilisation of inflammatory cells 27. The CX3CR1 gene is found on chromosome 3p21.3 and some of its genetic variants were discovered to promote AMD development 28, possibly through a loss of function in the receptor's activity. In fact, CX3CR1 proteins were found in a much lower concentration in the macular region of AMD individuals compared to non-affected subjects carrying the same polymorphism 29. This and other studies suggested that the diminished degree of inflammatory cells chemokinesis plays an important role in AMD 28, 29. Cx3cr1âˆ’/âˆ’ mice models exhibited a degeneration of the retina characterised by the accumulation of deposits containing microglia which share similar features with the typical AMD drusen 30. Microglia is the only cell in the human retina to carry CX3CR1 receptors 30. In addition to the importance of CX3CR1 receptors, these findings suggest a possible involvement of microglia deposits in the inflammation processes related to AMD.
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Many other inflammatory factors and receptors have been found to play a role in the pathogenesis of AMD. The polymorphic homozygotic variant A251T of the interleukin-8 (IL8) gene, for example, was described as a potential risk factor 31.
Moreover, the toll-like receptor (TLR) acts as important modulator in the innate immunity. Its defect function in the D299G variants may lead to an impaired microbial clearance, resulting in a predisposition to retinal inflammation and subsequent AMD development 2.
LOC387715/ARMS2 (age-related maculopathy susceptibility 2) and HtrA1 (high temperature required factor A-1)
Two large genome-wide linkage studies identified a locus on chromosome 10q26 as a possible candidate for the location of a common gene related to AMD 32, 33. This finding was confirmed by a genome-scan meta-analysis which involved six studies investigating more than 600 families in total 34. A subsequent single-nucleotide polymorphism study involving AMD families as well as a case-control study discovered the presence of a significant association with the disease of two genes in close proximity, i.e. LOC387715/ARMS2 and a heat shock serine protease known as HTRA1 35. Despite their near location, these genes appear to have a separate function within the retinal tissue 2.
The A69S polymorphism of ARMS2/LOC387715 has been linked to a higher susceptibility for AMD. Ross et al. reported that heterozygoty at this locus is related to odds ratio of 1.69-3.0 for severe AMD. Homozygote individuals for the A69S allele, on the other hand, produced an odds ratio of 2.20-12.1 36. In addition, the occurrence of this risk allele was found to be higher in subjects with late-stage AMD compared to the other less advanced stages 35.
The ARMS2 protein was discovered to be particularly expressed within the ellipsoid region of the retinal photoreceptors 37. Through the use of a particular marker, the specific location was then discovered to be the lumen of the mitochondria 38. These organelles appear to have an important role in the pathogenesis of AMD, decreasing in number and function in the photoreceptor and retinal pigment epithelium cells of subjects manifesting by this condition when compared to unaffected individuals 39.
Similarly, the HtrA1 gene which is in significant linkage disequilibrium with LOC387715 was discovered to possess four different SNPs which are associated with AMD. In particular rs11200638, which is localised in the gene's promoter region, has been reported to have an odds ratio of 1.60-2.61 in heterozygous and of 6.56-10.0 in homozygous subjects 2, confirming an evident link between the disease and this specific polymorphism.
In addition, the presence of either HtrA1 or the A69S SNPs of LOC387715 in smokers has been associated to an up to 5-fold enhanced risk of developing wet AMD compared to non-smokers 2. Nevertheless, this finding was recently challenged by another study which could not detect a higher risk in smokers carrying this specific allele's variant 40, despite validating the association between A69S and wet AMD.
HtrA1 belongs to the family of the heat shock serine proteases and is encoded in several cells of the human body including the retinal pigment epithelium 41. This protein was found to be highly expressed in the drusen and other pathological features typical of AMD 41.
Through an augmented production of matrix metalloproteinases, HtrA1 was discovered to play a role in the digestion of elements of the extracellular matrix, leading to chronic inflammation and neovascularisation 42. Its complex biological function, however, has not been fully elucidated yet 2.
Apolipoprotein E (ApoE)
Apo-E is an important component of various lipoproteins. Its main function relates to the transport of lipids into the cells' membranes, assisting the binding of low-density lipoproteins (LDL) to their corresponding receptors. This component is believed to have a role in the highly intensive synthesis of the membrane of the photoreceptors localised in the macula 43.
The gene encoding for ApoE is located on chromosome 19q13.2, and consists of three different polymorphic forms. While various studies reported a modification in the risk of developing AMD in the presence of different SNPs, other publications have detected no correlation 2.
ApoE was found in drusen of AMD patients 44 along with other lipids, and in particular cholesteryl esters and unsaturated fatty acids. It was speculated that the building up of these deposits may affect the barrier function of the Bruch's membrane, facilitating the formation of the macular disease 2.
Vascular endothelial growth factor (VEGF)
The members of the VEGF family, including VEGF-A, -B, -C, -D, -E, -F and placenta growth factor (PlGF), produce multiple pro-inflammatory reactions such as capillary leakage, angiogenesis, etc. In particular, VEGF-A plays an important role in AMD pathogenesis and was found in the macula of affected patients 45.
The gene encoding for VEGF-A is localised on chromosome 6p21 and consists of six different isoforms. The data regarding the association of VEGF-A and AMD are discordant. For example, in a case-control cohort study, the rs2010963 isoform was found to be linked to wet AMD 46. Similarly, both rs833070 and rs3025030 isoforms studied in a family cohort were found to be linked with the same macular disorder 46. Other studies, however, detected little or no association between the VEGF-A SNPs and different AMD forms.
Antibodies targeting VEGF-A are successfully used in clinical practice for the treatment of wet AMD 47. Despite the conflicting data on the association between VEGF-A and this macular disease, genetic testing may prove to be useful in identifying the candidate who could mostly profit from this kind therapy 2.
Age-related macular degeneration is a potentially blinding common disease of the elderly population. In the pathogenesis of this disorder a key role is played by the complement system and other inflammatory factors such as chemokines and VEGF. These elements have been discovered to lead to a chronic inflammatory state which causes damage of multiple retinal structures resulting in formation of drusen and potential neovascularisation.
Over most recent years, many genes as well as environmental elements have been identified as possible risk factors in the development of this complex disease. Further understanding of the role of the genes in AMD will probably allow to precisely targeting the management of patients according to their genetic profile and the environmental risk factors to which they have been exposed. In addition, a better knowledge of the mechanisms underlying this pathology will offer the basis for the creation of more effective treatments as well as preventive screening measures. This will reduce the actually high blinding rate caused by AMD, decreasing its social and economical burden.