WO2008077412A1

WO2008077412A1 - Heredity of small hard drusen and macular degeneration

Info

Publication number: WO2008077412A1
Application number: PCT/DK2007/050205
Authority: WO
Inventors: Lars Michael Larsen
Original assignee: Region Hovedstaden
Priority date: 2006-12-27
Filing date: 2007-12-27
Publication date: 2008-07-03

Abstract

The present invention relates to a method and a system for determining a predisposition for acquiring maculopathy and macular degeneration, including but not limited to age-related maculopathy and age-related macular degeneration, as well as data carrier comprising instructions for carrying out the methods, In particular it has been found that the presence of at least 6 small hard drusen in one eye is indicative of a predisposition for acquiring macula degeneration or maculopathy at a later stage in life.

Description

Heredity of small hard drusen and macular degeneration

AH patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to a method and a systemfor determining a predisposition for acquiring maculopathy and macular degeneration, including but not limited to age- related maculopathy and age-related macular degeneration, as well as data carrier comprising instructions for carrying out the methods,

Background of invention

Genetic variation, that is deviation from the most common and functionally intact form, such as the substitution of a single nucleotide, can have obvious deleterious consequences for the gene product and be a direct cause of disease, in which case the condition is called a mutation. Considerable variation exist in coding regions of the genome without the less frequent variants necessarily being associated with ciearly identifiable disease, in which case the condition is called a polymorphism. In age- related macular degeneration, such polymorphisms have been shown to be associated with an increased risk of age-related macular degeneration.

Small hard drusen are a common finding in healthy young and middle-aged subjects¹¹², but it is uncertain whether their presence predicts the development of symptomatic eye disease later in life and if, consequently, they offer an opportunity to monitor the effect of early intervention, e.g. the discontinuation of tobacco smoking or pharmaceutical interventions. It has been shown that large numbers of hard drusen predict the incidence of soft drusen and fundus pigmentation abnormalities, which are associated with an increased risk of developing of geographic atrophy and exudative maculopathy ^3;4. Development of age-related maculopathy (ARM) and age-related macular degeneration (AMD) is highly dependent on genetic factors. Notably, a study of 840 elderly male twins demonstrated a heritability of 46% for overall AMD grade and 71% for advanced AMD ⁵. The sequence variation Y402H in complement factor H has been shown to be related to an increased risk of age-related macular degeneration (Hageman GS et al.: A common hapiotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 2005;102:7227-7232. Klein RJ et al.: Complement factor H polymorphism in age- related macular degeneration. Science 2005;308:385-389. Edwards AO et al.: Complement factor H polymorphism and age-related macular degeneration. Science 2005;308:421-424. Haines JL et al.: Complement factor H variant increases the risk of age-related macular degeneration. Science 2005;308:419-421 ). Multiple polymorphisms serve alone or together to determine the risk of age-related macular degeneration, some haplotypes being associated with disease susceptibility and some with a protective effect (Li et a!.: CFH haplotypes without the Y402H coding variant show strong association with susceptibility to age-related macular degeneration. Nat Genet. 2006;38: 1049-1054.). Noncoding CFH variants also play a role in disease susceptibility {Malier et al.: Common variation in three genes, including a noncoding variant in CFH, strongly influences risk of age-related macular degeneration. Nat Genet. 2006:38:1055-1059). An additional risk factor has been found in the coding change (Ala69Ser) in the LOC387715 gene (Rivera A et al.: Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum MoI Genet.

2005; 14:3227-3236. Schmidt S: Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration, Am J Hum Genet. 2006;78:852- 864). Genetic variation in factor B and complement component 2, located in the major histocompatibility complex class III region, is associated with a risk hapiotype and two protective haplotypes. Combined analysis of the C2 and BF haplotypes and CFH variants showed that variation in the two loci predict the clinical outcome in 74% of the affected individuals and 56% of the controls (Gold B et al.: Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 2006;38:458-462). The summary conclusion of these AMD genotype studies is that while information about muitiple-loci genotypes and environmental risks can be combined to calculate an estimated risk that a given individual will develop age-related macular degeneration, the precision and accuracy of this estimate is likely to be limited. Depending upon the cost and the risks of preventive interventions, it may be difficult to make clinical decisions based upon genotype information alone. Consequently, there is reason to consider the clinical utility of early morphological markers of AMD-risk and risk of AMD-related visual loss.

Summary of invention

The present invention is based on the finding that the amount and/or distribution of small hard drusen appears to be genetically determined, and the inventors have found that even a small amount of small hard drusen and/or small hard drusen within a short distance of the macula lutea is indicative of a predisposition of acquiring macula degeneration. Due to the fact that the present inventors have been able to determine a lower limit of small hard drusen that is indicative of the predisposition of macula degeneration it has been possible for the present inventors to provide an automatic method and system for classifying retina images as images belonging to a person in risk of acquiring macula degeneration at a later stage in life, solely by determining the amount of small hard drusen and/or their distance to macula lutea, The risk of acquiring macula degeneration determined through image analysis may be supplemented with genotyptng of known risk genes.

The present invention is based on the finding that a genetic effect is detectable in the distribution of small hard drusen among twins, in particular in a population extending down to the age of 20 years, in particularly if imaging techniques are applied that facilitate the visibility of small hard drusen. In contrast to prior studies, the population did not include subjects with age-related macular degeneration, in contrast to a previous study, where a heritability of 81 % of the phenotype > 20 hard drusen per eye was found in 506 female twins pairs aged 49 to 79 years ⁶. In the present invention, the prevalence, systemic associations, concordance, and heritability of small hard drusen using red-free fundus photography in twins of both sexes aged 20 to 46 years have been examined.

The present inventors have found that an individual having at least 6 small hard drusen in one eye at a younger age, is a predisposition for acquiring macula degeneration at a later stage in life. By using such a diagnostic tool it is possible to prevent or reduce macula degeneration, for .example by advising the individuals of changing iife style. Accordingly, in one aspect the invention relates to a method for determining a predisposition for acquiring macula degeneration in an individual, comprising determining the number and/or position of small hard drusen in the retina of the individual, wherein a number of small hard drusen above 6 in at least one eye, and/or a position of small hard drusen within a predetermined distance to the macula is indicative of acquiring macula degeneration at a later stage in life.

In another aspect the invention relates to a method for determining a predisposition for acquiring macula degeneration in an individual, comprising

detecting in at least one retina image from said individual small hard drusen area(s), and

determining the number and/or position of said smail hard drusen area(s),

wherein a number of small hard drusen above 6 in at least one eye, and/or a position of small hard drusen within a predetermined distance to the macula is indicative of a predisposition for acquiring macula degeneration at a later stage in life.

In particular the determination of the small hard drusen may be carried out from one or more images of the retina. More preferred the invention relates to a method for determining the predisposition, wherein the method is carried out on images. Even more preferred the invention relates to an automatic grading of the images, so that it is only necessary for the physician to study images classified as being from an individual at risk.

Thus, in another aspect the invention relates to a method for classifying a fundus image comprising

- assessing the presence or absence of at least one small hard drusen area(s) by the method as defined above,

- grading the fundus image with respect to number and/or position of small hard drusen area(s), classifying the fundus image into at least two classes, wherein above 6 small hard drusen areas in at least one eye, and/or a position of small hard drusen within a predetermined distance to the macula leads to classification of said image as a an image of an individual at risk for acquiring macula degeneration at a later stage in life.

The method may be combined with a genetic method comprising the genotype of the individual and correlating the genotype to a risk genotype.

In another aspect the invention relates to a method for determining a predisposition for acquiring macula degeneration in an individual, comprising determining the genotype of the individual and comparing said genotype to the genotype of an individual suffering from small hard drusen in the retina, wherein polymorphism similarity in at ieast one gene is indicative of acquiring macula degeneration at a later stage in life.

The imaging methods according to the invention may also be used for monitoring an individual at risk as wel! as monitoring the efficacy of a treatment, and accordingly the present invention relates to a method for monitoring an individual with respect to macula degeneration, said method comprising

determining the number and/or position of said small hard drusen area(s),

comparing said number and/or position of said small hard drusen area(s) with at least one previous retina image from the same individual, and

determining whether the amount of small hard drusen and/or the position has changed.

The invention further relates to a system for carrying out the method of the present invention as well as a data carrier comprising instructions for carrying out the invention.

Accordingly, in a further aspect the invention relates to a system for determining a predisposition for acquiring macula degeneration in an individual, comprising an algorithm for detecting, in at least one image of retina of said individual, small hard drusen area(s), and

an algorithm for determining the number and/or position of said small hard drusen area(s),

an algorithm for providing information as to whether a number of small hard drusen exceeds 6 in at least one eye, and/or a position of small hard drusen is within a predetermined distance to the macula.

And in another aspect the invention relates to a system for classifying a fundus image comprising

an algorithm for assessing the presence or absence of at least one small hard drusen area(s) by the method as defined in claim 18,

- grading the fundus image with respect to number and/or position of small hard drusen area(s),

classifying the fundus image into at least two classes, wherein above 6 small hard drusen areas in at least one eye, and/or a position of small hard drusen within a predetermined distance to the macula leads to classification of said image as a an image of an individual at risk for acquiring macula degeneration at a later stage in life.

Furthermore, the invention relates to a data carrier for carrying out the methods described herein, as well as to a software program, both comprising instructions for carrying out the methods described herein.

Description of Drawings

Fig. 1. Montage of red-free fundus photographs representing macular distribution type of small hard drusen. In this eye, 75 small hard drusen were counted when allowing digital contrast enhancement and counting of every drusen anywhere in the fundus. Fig. 2. Montage of red-free fundus photographs representing small hard drusen of stippled, innumerable distribution type, with hundreds of drusen or drusen-like elements, ranging from such that are apparent at standard magnification and unaltered contrast to those that are distinguishable only after magnification and contrast enhancement (insert).

Fig. 3. Frequency distribution of small hard drusen in 116 monozygotic and 104 dizygotic healthy twins aged 20-46 y. The stippled fundus phenotype is illustrated in Fig. 2.

Fig. 4. The age-trajectory of the proportions p_; (x) of different drusen number categories (i=1 6 for <1 , 1-5, 6-10, 11-20, 21-40 and >40 drusen respectively), as predicted by the liability model, males (black lines), females (grey lines) . The line graphs correspond to the function f{x) = φ(t. + β_age ^■ x + β_sex ^■ sex), where β_age and β_s∞ ^are the regression coefficients describing the effects of age and gender with estimated values of -0.04 (Cl₉₅ -0.07,-0.02) and -0.34 (CI₉₅ -0.69, 0.01 ), respectively, t, are the liability thresholds and Φ is the cumulative distribution function of the standard normal distribution. Thus, the drusen category proportions are given as the vertical distances between the line graphs, corresponding to: ftW

+ β_aga -x + β_sex -sex)

Detailed description of the invention

Definitions

Age-related macular degeneration (AMD) is a medical condition in which the central retina (the macula) suffers from the abnormal accumulation of extracellular materia! (drusen). This condition is associated with loss of cells (atrophy, dry AMD) and/or formation of new vessels (neovascularization, wet AMD). All three processes have a deleterious effect on visual function. Treatment and preventive measures currently available are insufficient and AMD remains a prominent cause of severe visual loss.

Small hard drusen are tiny yellow or white accumulations of extracellular material that build up in Bruch's membrane of the eye Small hard drυsen area(s) are defined as any bright element of diameter less than 63 μm, the shape, color or proximity to adjacent pathology of which did not suggest that it could be hard exudate

Fovea: The term is used in its normal anatomical meaning, i.e. the spot in retina having a great concentration of rods giving rise to the vision. Fovea and the term "macula lutea" are used as synonyms.

Image: The term image is used to describe a representation of the region to be examined, i.e. the term image includes 1 -dimensional representations, 2-dimensional representations, 3-dimensiona!s representations as well as n-dimensional representatives. Thus, the term image includes a volume of the region, a matrix of the region as well as an array of information of the region.

Optic nerve head: The term is used in its normal anatomical meaning, i.e. the area in the fundus of the eye where the optic nerve enters the retina. Synonyms for the area are, for example, the "blind" spot, the papula, or the optic disc.

As used herein, "allele", which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When an individual has two identical alleles of a gene, the individual is said to be homozygous for the gene or allele. When an individual has two different alleles of a gene, the individual is said to be heterozygous for the gene or alleles. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.

As used herein, "predisposition" means that an individual having a particular genotype and/or haplotype has a higher likelihood than one not having such a genotype and/or haplotype for a particular condition/disease as one of the described herein.

As used herein, the term "haplotype" refers to a set of closely jinked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination). Some haplotypes may be in linkage disequilibrium. As used herein, the term "genetic marker^' refers to an identifiable physical location on a chromosome (e.g., single nucleotide polymorphism (SNP), restriction enzyme cutting site) whose inheritance can be monitored. Markers can be expressed regions of DNA (genes) or some segment of DNA with no known coding function but whose pattern of inheritance can be determined.

As used herein, the term "linkage" refers to an association in inheritance between genetic markers such that the parental genetic marker combinations appear among the progeny more often than the non-parental.

As used herein, the term "linkage disequilibrium" (LD) means that the observed frequencies of haplotypes in a population does not agree with hapiotype frequencies predicted by multiplying the frequencies of individual genetic markers in each hapiotype; LD means that there exist correlations among neighbouring alleles, reflecting 'haplotypes' descended from single, ancestral chromosomes.

Predisposition to acquired macula degeneration

As described above macula degeneration and maculopathy, such as age-related macula degeneration and age-related maculopathy, is a severe disease of the eye. It is an advantage to predict the individuals in risk of acquiring the disease in order to prevent the disease or reduce the symptoms of the disease. Due to the present invention it is possible to classify image(s) of retina as image(s) belonging to an individual at risk of acquiring the disease. It has been made possible due to the fact that the present inventors have found a lower limit of the amount of small hard drusen in the retina and/or the position of said smal! hard drusen in relation to the macula lutea that indicates a predisposition to the disease, whereby an automatic method and system may classify the images, thereby assisting the physician.

The present inventors have found that the appearance of at least 6 small hard drusen in one eye is indicative of a predisposition of acquiring macula degeneration at a later stage in life. It has also been confirmed that the higher number of smal! hard drusen the higher risk. Thus, in one embodiment the number of small hard drusen in one eye is at least 8, such as 10 or more, such as 15 or more, such as 20 or more, such as 25 or more, such as 30 or more, such as 35 or more, such as 40 or more, in order to be indicative of the predisposition.

Accordingly, the retina image(s) may be classified as belonging to an individual having at least 6 small hard drusen in one eye, such as at least 8, such as 10 or more, such as 15 or more, such as 20 or more, such as 25 or more, such as 30 or more, such as 35 or more, such as 40 or more, small hard drusen.

In another embodiment the image(s) may be classified as belonging to any of the following categories; <1 , 1-5, 6-10, 11-20, 21-20, or >40, small hard drusen in one eye. Again the categories having at least 6 small hard drusen in one eye, are the categories of individuals at risk of acquiring macula degeneration at a later stage.

As an alternative, or in addition to the number of small hard drusen, the position of the small hard drusen is also indicative of the predisposition of acquiring macula degeneration. It has been found that the closer to the macula lutea the greater the risk.

Thus, in a preferred embodiment the image(s) are classified as belonging to an individual at risk of acquiring macula degeneration if the distance between a small hard druse and macula iutea corresponds to the distance between macula and the optic nerve or less, such as a distance corresponding to 7a the distance between macula and the optic nerve head or iess, such as a distance corresponding to 1/4 the distance between macula and the optic nerve head or less.

In particular the method according to the present invention is useful for predicting the risk of acquiring the later disease in individuals being in the age range of from 20 to 46 years, such as in the age range of from 20 to 35 years, at the time for the acquiring of the retina images.

Images

Fundus image is a conventional tool for examining retina and may be recorded on any suitable means. In one embodiment the image is presented on a medium selected from dias, paper photos or digital photos. However, the image may be any other kind of representation, such as a presentation on an array of elements, for example a CCD. The image may be a grey-toned image or a colour image; it is however preferred that the image is a red-free image, such as a grey-toned image. The images are preferably obtained using a red-free illumination.

One image per eye may be acquired, it is however preferred, to obtain several images per eye, such as four images per eye, whereby a better coverage of retina is obtained.

Identification of small hard drusen area(s)

The small hard drusen area(s) are preferably identified are extremas in the image, such as local extrema. The extrema may be minima or maxima or both.

The extrema may be identified in an unfiltered image, it is however preferred that the image is a filtered image, wherein the filtering may be linear and/or non-linear. It is within the scope of the invention, that the image may be filtered with one or more filters before identifying the extremas, or as a part of the step of identifying the extremas. Thus, in one embodiment of the invention the extremas are identified by combining two or more filters.

In one embodiment the filtering method is a template matching method, wherein the template may exhibit any suitable geometry for identifying the small hard drusen areas. Examples of templates are circles, wherein the circles have a radius set as a ratio of the expected diameter of the optic nerve head or as a ratio as the expected ratio of a small hard drusen area.

The extrema may thus be identified indidually by one or more of several methods, such as the following:

The small hard drusen areas are normally either dark areas or light areas in the image, or at least locally the darkest areas or the lightest areas. Thus, a method may be establishing at least one intensity extremum in the image, preferably at least one intensity minimum or at least one intensity maximum. Therefore, in a preferred embodiment at least one loca! intensity maximum is established. The extrema may be established on any image function, such as wherein the image function is the unsharp image, the green channel image, or any combinations thereof. The extrema may be corrected with respect to the background variation.

Instead of using intensity or in addition to using intensity the method may include establishing at least one variance extremum in the image, preferably establishing at least one variance maximum in the image. For the same reasons as described with respect to the intensity at least one local variance maximum is established. The extrema may be established on any image function, such as wherein the image function is the unsharp image, the green channel image, or any combinations thereof.

Once the small hard drusen area(s) have been identified, the amount of small hard drusen per eye is calculated. If one image of the retina, either acquired as one image or stitched together from several images, is acquired for each eye, then the number of small hard drusen area(s) in said image is calculated. If more separate images are acquired per retina, then the number of small hard drusen area(s) in the total of the images minus small hard drusen imaged in more than one image, so that the total sum of small hard drusen area(s) includes only once any small hard drusen imaged on two or more images of the same eye.

Furthermore, or alternatively, the distance of each small hard drusen area to the macula lutea is determined, for example as a ratio of the distance between the optic nerve head and the macula lutea. The shortest distance between a small hard drusen area and the macula lutea determined is preferably selected as the distance determining the classification of the image(s) of said individual. For example if 7 small hard drusen areas are determined one eye, then the small hard drusen area for said eye having the shortest distance to the macula lutea determines the classification, independent of whether all the remaining 6 small hard drusen areas have a much longer distance. In another embodiment the predetermined distance is a distance calculated as an average of the distance of at least 3 small hard drusen areas in the image(s) of one eye, such as an average of at least 5 small hard drusen areas.

Grading

The images may then be.graded into at least two categories, said categories being individuals not being at risk or individuals being at risk, wherein the determination of categories are determined as described above with respect to number and/or position of small hard druseπ in at least one eye.

The images may be graded into more than two categories, such as for example three categories being "no risk >6, medium risk 6-20, high risk >20", wherein the number relates to the number of small hard drusen area(s) in one eye, or for example the categories described above, namely <1, 1-5, 6-10, 11-20, 21-20, or >40 small hard drusen in one eye, whereby the classification may be more differentiated.

As described below, the determination of a predisposition for acquiring macula degeneration conducted according to this invention may be supplemented with a genotyping for genes and polymorphisms known to be indicative for the predisposition as well. Thus, the method may further include parameters from a genotyping or any other type of parameter known to be indicative for the predisposition as well, see table 4 for parameters normally assessed.

Monitoring

The imaging methods according to the invention may also be used for monitoring an individual at risk as well as monitoring the efficacy of a treatment. For example an individual being found at risk of developing macula degeneration may be examined every year or every second year by means of retina images analysed as described herein. A progression in the condition may lead to reconsideration of treatment and/or change in lifestyle.

System

The methods according to the present invention are preferably carried out on a system as described below, and accordingly, the invention further relates to a system for carrying out the methods.

The system comprises at least a processor capable of performing the calculations, and preferably also a display screen capable of displaying the results of the calculations, such as the classification, and preferably also capable of displaying the images with a marking of the small hard drusen areas determined in the image. Pre-acquired image data can be fed directly into the system through a network interface and stored locally on a mass storage device and/or in a memory. Furthermore, image data may also be supplied over a network, through a portable mass storage medium such as a removable hard disk, optical disks, tape drives, or any other type of data transfer and/or storage devices which are known in the art.

in another embodiment the image data may be forwarded to a central computer, and the final results may be displayed at a display at the location of the treating physician.

One skilled in the art will recognize that a parallel computer platform having multiple processors is also a suitable hardware platform for use with a system according to the present invention. Such a configuration may include, but not be limited to, parallel machines and workstations with multiple processors. The processing system can be a single computer, or several computers can be connected through a communications network to create a logical processing system.

The present system allows the grader, that is the person normally grading the images, to identify the small hard drusen areas more rapidly and securely. Also, the present system allows an automatic detection of small hard drusen areas of the retina as an aiding tool for the traditional grader.

By use of the present system it is also possible to arrange for recordation of the images at one location and examining them at another location. For example the images may be recorded by any optician or physician or elsewhere and be transported to the examining specialist, either as photos or the like or on digital media. Accordingly, by use of the present system the need for decentral centers for recording the image, while the maintaining fewer expert graders could be realised.

Furthermore, in addition to the communication of images and medical information between persons involved in the procedure, the network may carry data signals including control or image adjustment signals by which the expert examining the images at the examining unit directly controls the image acquisition occurring at the recordation localisation, Ke. the acquisition unit. In particular, such command signals as zoom magnification, steering adjustments, and wavelength of field illumination may be selectively varied remotely to achieve desired imaging effect. Thus, questionable tissue structures requiring greater magnification or a different perspective for their elucidation may be quickly resolved without ambiguity by varying such control parameters. Furthermore, by switching illumination wavelengths views may be selectively taken to represent different iayers of tissue, or to accentuate imaging of the vasculature and blood flow characteristics. The digital data signals for these operations may be interfaced to the ophthalmic equipment in a relatively straightforward fashion, provided such equipment already has initiating switches or internal digital circuitry for controlling the particular parameters involved, or is capable of readily adapting electric controls to such control parameters as system focus, illumination and the like.

Thus, a very precise and well-annotated medical record may be readily compiled and may be compared to a previously taken view for detailed evidence of changes over a period of time, or may be compared, for example, to immediately preceding angiographic views in order to assess the actual degree of blood flow occurring therein. As with the ophthalmologist's note pad entries at examination unit, the measurement entries at examination unit become an annotated image record and are stored in the central library as part of the patient's record.

Unlike a simple medical record system, the present invention changes the dynamics of patient access to care, and the efficiency of delivery of ophthalmic expertise in a manner that solves an enormous current health care dilemma. A basic embodiment of the invention being thus disclosed and described, further variations and modifications will occur to those skilled in the art, and all such variations and modifications are encompassed within the scope of the invention as defined in the claims appended hereto.

In another aspect, the present invention relates to a computer readable medium comprising instructions for carrying out at least one of the methods according to the present invention as well as a computer program comprising instructions for carrying out at least one of the methods according to the present invention.

Methods of determining polymorphisms indicative for macula degeneration As described above, the methods according to this invention may be supplemented with geπotypiπg methods capable of determining genotypes indicative for a predisposition to the disease. Such genotyping may be carried out on DNA, RNA or proteins as described below.

SNP

Many methods (see Table 1 below) are known in the prior art for determining the presence of particular nucleotide sequences or for determining particular proteins having particular amino acid sequences. Ail of these methods may be adapted for determining the polymorphisms according to the present invention. Table !

One common method for detecting SNPs comprises the use of a probe bound to a detectable label. By carrying out hybridisation under conditions of high stringency it is ensured that the probe only hybridises to a sequence which is 100% complementary to the probe.

According to the present invention this method comprises hybridising a probe to a target nucleic acid sequence comprising at least one of the SNPs at the positions identified in genes of an individual being at risk for acquiring or having acquired macula degeneration (see above). In particular said genes are selected from genes of the complement system or in a chromosome region containing said gene.

In one embodiment the at least one polymorphism is determined in a gene selected from complement factor H gene, LOC387715 gene, factor B gene and/or complement component 2 gene or chromosome regions containing said genes. Preferably, at least one of the polymorphisms is determined in a non-coding region of a gene such as an intron or in a region controlling expression of the gene.

In one embodiment the at least one of the polymorphisms is determined in a non- coding region of the complement factor H gene, LOC387715 gene, factor B gene and/or complement component 2 gene such as an intron region.

In another embodiment the at least one polymorphism is determined in the region comprising a nucleotide sequence controlling expression of complement factor H gene, LOC387715 gene, factor B gene and/or complement component 2 gene, such as a promotor region.

For other polymorphisms or mutations within the defined region, similar probes can be designed by the skilled practitioner and used for hybridisation to a target nucleic acid sequence. The design and optimisation of probes and hybridisation conditions lies within the capabilities of the skilled practitioner. In the scope of the present invention the term "hybridisation" signifies hybridisation under conventional hybridising conditions, preferably under stringent conditions, as described for example in Sambrook et a!., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The term "stringent" when used in conjunction with hybridisation conditions is as defined in the art, i.e. 15-20⁰C under the melting point T_m, cf. Sambrook et af, 1989, pages 11.45-11.49. Preferably, the conditions are "highly stringent", i.e. 5-10⁰C under the melting point T_m. Under highly stringent conditions hybridisation only occurs if the identity between the oligonucleotide sequence and the locus of interest is 100 %, while no hybridisation occurs if there is just one mismatch between oligonucleotide and DNA locus. Such optimised hybridisation results are reached by adjusting the temperature and/or the ionic strength of the hybridisation buffer as described in the art. However, equally high specificity may be obtained using high-affinity DNA analogues. One such high-affinity DNA analogues has been termed "locked nucleic acid" (LNA). LNA is a novel class of btcyclic nucleic acid analogues in which the furanose ring conformation is restricted in by a methylene linker that connects the 2-0 position to the 4'-C position. Common to ati of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analogue (ørum et al. (1999) Clinical Chemistry 45, 1898-1905; WO 99/14226 EXIQON). LNA probes are commercially available from Proligo LLC, Boulder, Colorado, USA. Another high-affinity DNA analogue is the so-called protein nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylgiycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone {Science (1991) 254: 1497-1500).

Various different labels can be coupled to the probe. Among these fluorescent reporter groups are preferred because they result in a high signal/noise ratio.

Suitable examples of the fluorescent group include fluorescein, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, acridin, Hoechst 33258, Rhodamine, Rhodamine Green, Tetramethylrhodamine, Texas Red, Cascade Blue, Oregon Green, Alexa Fluor, europium and samarium. Another type of labels are enzyme tags. After hybridisation to the target nucleic acid sequence a substrate for the enzyme is added and the formation of a coloured product is measured. Examples of enzyme tags include a beta-Galactosidase, a peroxidase, horseradish peroxidase, a urease, a glycosidase, alkaline phosphatase, chloramphenicol acetyltransferase and a luciferase.

A further group of labels include chemϋuminescent group, such as hydrazides such as luminol and oxalate esters.

A still further possibility is to use a radioisotope and detect the hybrid using scintillation counting. The radioisotope may be selected from the group consisting of ³²P, ³³P, ³⁵S, ¹²⁵1, ⁴⁵Ca, ¹⁴C and ³H.

One particularly preferred embodiment of the probe based detection comprises the use of a capture probe for capturing a target nucleic acid sequence. The capture probe is bound to a solid surface such as a bead, a well or a stick. The captured target nucleic acid sequence can then be contacted with the detection probe under conditions of high stringency and the allele be detected.

One embodiment of the probe based technique based on TAQMAN technique. This is a method for measuring PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TAQMAN® probe. This probe is composed of a short (ca. 20-25 bases) oligodeoxynucleotide that is labeled with two different fiourescent dyes. On the 5' terminus is a reporter dye and on the 3' terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR ampiicon. When the probe is intact, energy transfer occurs between the two flourophors and emission from the reporter is quenched by the quencher. During the extension phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymerase thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity.

Other suitable methods include using mass spectrometry, single base extension, determining the Tm profile of a hybrid between a probe and a target nucleic acid sequence, using single strand conformation polymorphism, using single strand conformation polymorphism heteroduplex, using RFLP or RAPD, using HPLC, using sequencing of a target nucleic acid sequence from said biological sample.

Denaturing high-performance liquid chromatography (DHPLC) has been proven useful in human and animai genetic studies for detecting single nucleotide polymorphisms

(SNPs). In contrary to most SNP detection methods that are currently in used, SNP detection by DHPLC is not based on a re-sequencing strategy that is expensive to implement, nor does it require gel-based genotyping procedures. Instead, SNP detection by DHPLC is based on resolving heteroduplex from homoduplex DNA fragments produced by PCR amplification using temperature-modulated heteroduplex analysis.

In connection with several of these methods there is a need for amplifying the amount of target nucleic acid in the biological sample isolated from the subject. Amplification may be performed by any known method including methods selected from the group consisting of polymerase chain reaction (PCR)₁ Ligase Chain Reaction (LCR)₁ Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification, rolling circle amplification, and T7-polymerase amplification.

One of the primers may comprise a moiety for subsequent immobilisation of the amplified fragments.

It is understood that the primers identified above may also be used as probes for determining the polymorphisms of the invention in a nucleic acid sequence using any of the methods known in the art and featured above.

To the extent that the polymorphisms as defined in the present invention are present in DNA sequences transcribed as mRNA transcripts these transcripts constitute a suitable target sequence for detection of the polymorphisms. Commercial protocols are available for isolation of total mRNA. Through the use of suitable primers the target mRNA can be amplified and the presence or absence of polymorphisms be detected with any of the techniques described above for detection of polymorphisms in a DNA sequence.

Proteins Genetic polymorphism can also be detected as a polymorphism of a protein product of the gene, or a change in a biological response, e. g. immune response, where the protein is involved.

For example, the genetic polymorphisms according to the present invention may influence the co-stimulatory signalling in T cell activation or are linked to polymorphisms having this physiological effect, the diagnosis may also be carried out by measuring the relative amount of cytokines expressed downstream from the co- stimulatory signal in immune response pathway in a biological sample from a subject suffering from said diseases.

Polymorphism of a gene of the invention may also be identified by using an antibody raised against a variant protein expressed by the polymorphic gene. By using an antibody which is able to recognise an epitope comprising a region of the variant protein comprising a polymorphism corresponding to the polymorphism of the gene it is possible to determine a predisposition of an individual to macula degeneration of the invention without screening the genetic material. Thus, an antibody which is capable of specifically binding to an epitope comprising a polymorphism of the invention is also in the scope of the invention.

Antibodies within the invention include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab¹ fragments, F(ab')₂ fragments, and molecules produced using a Fab expression library, and antibodies or fragments produced by phage display techniques.

Polyclonal and/or monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using variant proteins (natural or recombinant) or fragment of these proteins which contain the polymorphism by standard technologies.

In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al_M Nature 256:495, 1975, and U.S. Patent No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole et ai., Proc. Natl. Acad. ScL USA 80:2026, 1983), and the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc., pp. 77- 96, 1983), Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE₁ IgA, IgD and any subclass thereof. (In the case of chckens, the immunoglobulin class can also be IgY.) The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo, The ability to produce high titers of mAbs in vivo makes this the presently preferred method of production, but in some cases, in vitro production will be preferred to avoid introducing cancer cells into ϋve animais, for example, in cases where the presence of normal immunoglobulins coming from the acitis fluids are unwanted, or in cases involving ethical considerations.

Once produced, polyclonal, monoclonal, or phage-derived antibodies are tested for specific recognition of the above described epitope by Western blot or immuno- precipitation in samples containing the polypeptides comprising the binding site or fragments thereof, e.g., as described in Ausubel et al., supra. Antibodies that specifically recognise a polymorphism of the variant protein are useful in the invention. Such antibodies can be used in an immunoassay to monitor the spectrum of the expressed protein of lnterst or a level of expression a variant protein in a sample collected from an individual. An antibody with is capable to inhibit an immune related activity of a variant protein is of a particular interest as a candidate compound for the treatment of macula degeneration of the invention.

The antibody may aiso be used in a screening assay for measuring activity of a polymorphic gene of the invention, for example as a part of a diagnostic assay. Depending on the detection technique the antibody may be coupled to a compound comprising a detectable marker. The markers or labels may be selected from any markers and labels known in the art. The antibody may also be used for determining the concentration of a substance comprising an epitope or epitope in a solution of said substance or said epitope. A wide spectrum of detection and labelling techniques is available now in the art and the techniques may therefore be selected depending on skills of the artisan practising the antibodies or on the purpose of using thereof.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA₁ 81 :6851 , 1984; Neuberger et al., Nature, 312:604,

1984; Takeda et al., Nature, 314:452, 1984) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778, 4,946,778, and 4,704,692) can be adapted to produce single chain antibodies against a variant protein of the invention or a fragment thereof comprising a polymorphim. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognise and bind to specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule, and Fab' fragments that can be generated by reducing the disulfide bridges of F(ab')₂ fragments. Alternatively, Fab' expression libraries can be constructed (Huse et al., Science, 246:1275, 1989) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.

Antibodies can be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). Fully human antibodies, such as those expressed in transgenic animals are also features of the invention (Green et al., Nature Genetics 7:13-21 , 1994; see also U.S. Patents 5,545,806 and 5,569,825, both of which are hereby incorporated by reference).

Thus, isolated/identified variant proteins expressed by any of the other polymorphic genes of the invention may be used as alternative diagnostic markers of the genetic polymorphism associated with a predisposition to macula degeneration of the invention.

Biological sample

The biological sample used for genotyping in the present invention may be any suitable biological sample comprising genetic material and/or proteins involved in induction of the immune response as described previously. In a preferred embodiment the sample is a blood sample, a tissue sample, a secretion sample, semen, ovum, hairs, nails, tears, and urine. The most convenient sample type is a blood sample.

Isolated oligonucleotides

In one aspect the invention relates to an isolated oligonucleotide comprising at least 10 contiguous nucleotides being 100% identical to a subsequence of the genes of the invention comprising or adjacent to a polymorphism or mutation being correlated to macula degeneration, or being 100% identical to a subsequence of the human genome which is in linkage disequilibrium with any of the genes of the invention comprising or adjacent to a polymorphism or mutation being correlated to macula degeneration. As explained in the summary, such probes may be used for detecting the presence of a polymorphism of interest and/or they may constitute part of a primer pair and/or they may form part of a gene therapy vector used for treating the macula degeneration.

The length of the isolated oligonucleotide depends on the purpose. When being used for amplification from a sample of genomic DNA, the length of the primers should be at least 15 and more preferably even longer to ensure specific amplification of the desired target nucleotide sequence. When being used for amplification from mRNA the length of the primers can be shorter while still ensuring specific amplification. In one particular embodiment one of the pair of primers may be an allele specific primer in which case amplification only occurs if the specific allele is present in the sampie. When the isolated oligonucleotides are used as hybridisation probes for detection, the length is preferably in the range of 10-15 nucleotides. This is enough to ensure specific hybridisation in a sample with an amplified target nucleic acid sequence. When using nucleotides which bind stronger than DNA (e.g. LNA and/or PNA), the length of the probe can be somewhat shorter, e.g. down to 7-8 bases.

The length may be at least 15 contiguous nucleotides, such as at least 20 nucleotides. An upper limit preferably determines the maximum length of the isolated oligonucleotide. Accordingly, the isolated oligonucleotide may be less than 1000 nucleotides, more preferably less than 500 nucleotides, more preferably less than 100 nucleotides, such as less than 75 nucleotides, for example less than 50 nucleotides, such as iess than 40 nucleotides, for example less than 30 nucleotides, such as less than 20 nucleotides. The isolated oligonucleotide may comprise from 10 to 50 nucleotides, such as from 10 to 15, from 15 to 20, from 20 to 25, or comprising from 20 to 30 nucleotides, or from 15 to 25 nucleotides.

Depending on the use the polymorphism may be located in the centre of the nucleic acid sequence, in the 5' end of the nucleic acid sequence, or in the 3' end of the nucleic acid sequence.

For detection based on single base extension the sequence of the oligonucleotide is adjacent to the mutation/polymorphism, either in the 3' or 5' direction.

The isolated oligonucfeotide sequence may be complementary to a sub-sequence of the coding strand of a target nucleotide sequence or to a sub-sequence to the non- coding strand of a target nucleotide sequence as the polymorphism may be assessed with similar efficiency in the coding and the non-coding strand.

The isolated oligonucleotide sequence may be made from RNA, DNA, LNA₁ PNA monomers or from chemically modified nucleotides capable of hybridising to a target nucleic acid sequence. The oligonucleotides may also be made from mixtures of said monomers.

Kits

In one aspect there is provided a kit for predicting the risk of a subject for developing immune related diseases or for other diagnostic and classification purposes of immune related diseases comprising at least one probe comprising a nucleic acid sequence as defined in the previous section.

In one embodiment the probe is linked to a detectable label.

In another embodiment based on single nucleotide extension the kit further comprises at least one nucleotide monomer labelled with a detectable label, a polymerase and suitable buffers and reagents. A kit may also comprise an antibody capable of recognising the poiumorphism of the invention.

Treatment

Once an individual has been classified as an individual at risk of acquiring macula degeneration it is relevant to discuss prophylactic treatment and change of life style. Change of lifestyle may be giving up smoking, intake of non-fat or low-fat diets, and starting exercising.

Furthermore, a more specific therapy may be gene therapy as described below.

Gene therapy

Having identified polymorphism(s) as the cause of a disease it is also rendered possible with the present invention to provide a genetic therapy for subjects being diagnosed as having a predisposition according to the invention, said therapy comprising administering to said subject a therapeutically effective amount of a gene therapy vector. The gene therapy vectors carry the protective allele of the genes. The protective allele means in the present content that expression of this allele in an individual indicates no predisposition to macula degeneration of the invention.

There are various different methods of gene therapy for the subjects defined in the present invention.

The first two are based on activation of the repair system of the cells by introducing into those cells a gene therapy vector which causes "correction" of the polymorphism by presenting the repair mechanism with a template for carrying out the correction. One such type includes the RNA/DNA chimeraplast, said chimeraplast being capable of correcting the polymorphism in cells of said subject. Examples of the design of such chimeraplasts can be found in e.g. US 5,760,012; US 5,888,983; US 5,731 ,181 ; US 6,010,970; US 6,211,351.

The second method is based on application of single stranded oligonucleotides, wherein the terminal nucleotides is protected from degradation by using 3¹ and 5' phosphorothioat-linkage of the monomers. This gene therapy vector is aiso capable of "correcting" the polymorphism by replacing one nucleotide with another. These first two types of gene therapy vectors comprise a small sequence (less than 50 bases) which overlaps with the polymorphism in question. Suitable sequences for this purpose are genomic sequences located around the polymorphism.

Other types of gene therapy include the use of retrovirus (RNA-virus). Retrovirus can be used to target many ceils and integrate stably into the genome. Adenovirus and adeno-associated virus can also be used. A suitable retrovirus or adenovirus for this purpose comprises an expression construct with the wϋdtype gene under the contro! of the wildtype promoter or a constitutive promoter or a regulatabie promoter such as a repressible and/or inducible promoter or a promoter comprising both repressible and inducible elements.

A further group of gene therapy vectors includes vectors comprising interfering RNA (RNAi) for catalytic breakdown of mRNA carrying the polymorphism. RNAi can be used for lowering the expression of a given gene for a relatively short period of time. In particular these RNAi oligos may be used for therapy for both subjects carrying a susceptibility allele as described in the present invention as well as for subjects which do not carry such an allele.

Interfering RNA ("RNAi") is double stranded RNA that results in catalytic degradation of specific mRNAs, and can also be used to lower gene expression.

When the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extraceiiular environment, does not allow the introduced cells to be recognized by the host immune system.

Other therapies

The subjects carrying the mutations as defined in the present invention may also be treated using conventional therapy. The principles behind conventional therapy are described in short below. Drug discovery

Once an individual has been diagnosed as having a predisposition for the disease, it may be considered to use his or her genotype as a model in drug discovery process.

Accordingly, a cell line based on cells isolated from a subject carrying a polymorphism according to the invention may aiso be cultured and used for the screening purposes.

The vector may comprise part(s) of the nucleotide sequence discussed above, said sequence comprising a polymorphism associated with macula degeneration. Using this vector more precisely mimics the expression in vivo due to the presence of introns and possibly the native promoter of the genes.

According to some embodiments the vector may comprise a constitutive promoter. According to other embodiments the vector may comprise a promoter sequence comprising a reguiatable promoter such as a viral promoter sequence.

The vector may be transferred into a host ceil which can be used for screening purposes in drug discovery. The host cells may be selected from a bacterial cell, a yeast cell, a mammalian cell line, more preferably a human celi line. More preferably, the host ceil is a human immortalised celi line such as human melanocyte.

Screening of compounds for a functionality related to macula degeneration can be carried out by exposing a cell as described above to a drug candidate and measuring a response related to the co-stimulatory signal.

One method that detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described {Chien, et al., 1991 , Proc. Natl. Acad. Sci. USA, 88, 9578- 9582) and is commercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the gene peptide product of the genes mentioned above and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.

Pharmaceutical composition Once the candidate compound(s) of the invention has been identified by the drug discovery method it is further within the scope of the invention to provide a pharmaceutical composition comprising one or more compound(s). In the present context the term pharmaceutical composition is used synonymously with the term medicament.

Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art. The formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect. Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonai and, in some cases, oral formulations. For suppositories, traditional binders and carriers include polyalkyiene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonai administration, e.g. iπhalators and aerosols.

The active compound may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethy!amino ethanol, histidiπe, procaine, and the like.

The preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 5000 μg per kilo body weight. Using monomeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 5000 μg per kilo body weight, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 1000 μg per kilo body weight, such as in the range of from about 0.1 μg to 750 μg per kilo body weight, and especially in the range of from about 0.1 μg to 500 μg per kilo body weight such as in the range of from about 0.1 μg to 250 μg per kilo body weight. In particular, when administering nasally smaller dosages are used than when administering by other routes. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body weight. For some indications a localised or substantially localised application is preferred.

For other indications, intranasal application is preferred.

Some of the compounds of the present invention are sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it wiil be advantageous to include a compound, which promotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc. it is preferred that administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to development of an immune related disease of the invention. Preferably the medicament is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.

In another aspect the invention relates to a process of producing a pharmaceutical composition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers, and administer an effective amount of at least one of said compound, or said pharmaceutical composition to a subject.

In yet a further aspect the invention relates to a method of treating an individual suffering from one or more of the diseases discussed above by administering the said individual a compound as described herein or a pharmaceutical composition comprising said compound.

Example 1 Methods; subjects and protocol: We examined 59 monozygotic (MZ) and 55 dizygotic (DZ) same-sex twin pairs aged 20 to 46 years. The subjects were recruited from the population-based Danish Twin Registry⁷ which includes more than 65000 twin pairs in birth cohorts from 1870 to 1996, the ascertainment rate being 90 % up to 1968 and 100% after 1968, when a fully comprehensive computerized national population database was introduced (Danish Civil Registry), Zygosity was determined using nine polymorphic DNA-based microsatellite markers (AmpFiSTR Profiler Plus Kit; Perkin Eimer Applied Biosystems, Foster City, CA₁ USA). This principle has an error probability of 0.003% or lower ⁸. The twins participated in a larger study of diabetes- related metabolism ⁹ for which participants were invited using a mailed questionnaire. Exclusion criteria were: pregnancy, breastfeeding, known diabetes or cardiovascular disease and conditions preventing the completion an ergometric bicycle test. Of 2099 invited pairs, 764 pairs were eligible and willing to participate. Randomized exclusions were made in specific age groups to achieve a uniform age distribution, reducing the participant number to 621. Of these, twin pairs where both twins lived on the island of Zeeland (Sjaslland) were invited to participate in a separate ophthalmic examination, for which 114 pairs volunteered. Previously published data include lens autofluorescence ¹⁰, retinal nerve fiber layer thickness ¹¹, and the presence of cilioretinal arteries ¹². The present study excluded 1 MZ twin pair and 3 DZ twin pairs because fundus photography was unavailable on the day of examination. Thus, the study included 58 MZ and 52 DZ twin pairs.

All participants gave their informed consent. The study was approved by the regional medicai ethics committee and followed the tenets of the Helsinki Declaration. Subjects responded to a detailed questionnaire including information about lifelong smoking habits. Study examinations that previously have been described in detail^9"12 included oral glucose tolerance testing, blood pressure measurement, blood sampling, and measurement of height and weight. The ophthalmic examination included refractioning, determination of Snellen visual acuity, pupil dilation, slit-lamp biomicroscopy, non-stereoscopic digital greyscale fundus photography on a 1024x1024 pixel image sensor, four 50 degree fields per eye and a single 20 degree optic disk- centered photograph, in red-free illumination (Wratten 54 filter; Eastman Kodak, Inc., Rochester, NY), and color diapositive photography (four 50-degree fields per eye). Methods; fundus morphology grading: Fundus characteristics were visually assessed by examination of digital images on a computer screen and by diapositive inspection using a hand-held 15 D lens. Histogram stretching was allowed during the evaluation of the digital photographs. Small hard drusen were defined as any bright element of diameter less than 63 μm, the shape, color or proximity to adjacent pathology of which did not suggest that it could be hard exudate. Specific attention was also given to the differentia! diagnosis of small hard drusen versus subretinal precipitate and multifocal retinal pigment epithelium depigmentation secondary to central serous chorioretinopathy ¹³. Drusen associated with a nevus were not included. A single observer examined all greyscale photographs, in random order and masked to the zygosity and relatedness of the subjects. Every drusen was counted and marked, regardless of location in the fundus except in subjects with stippling where the drusen were too numerous and too difficult to discern from the background for exact counting. When small hard drusen areas were deemed questionable or other retinal pathology was present, the color diapositives were evaluated and a second observer consulted. After the first round of examination, the observer was able to identify 3 patterns of distribution of drusen. The photographs were re-evaluated twice and subjects with 5 or more drusen per eye (mean of the subject's two eyes) could consistently be classified into 3 categories: I) scattered drusen, Ii) macular drusen (Fig. 1), III) stippled fundus with innumerable drusen (Fig, 2). The latter category was characterized by subtle stippling that appeared to represent minute drusen, ranging from definitely visible small hard drusen to smaller elements of the same type but visible only after stretching of the luminosity range of the photograph. Manual counting of selected fundus areas and subsequent extrapolation to the entire combination of photographic field yielded estimates in excess of 1000 elements per eye. This phenotype has previously been described ¹⁴. Methods; statistical procedures: The classical twin data analysis is based on the assumption that MZ twins have identical genotypes, for which reason all observed differences between twins in a pair are attributable to environmental factors. Dizygotic twins, however, share on average 50% of their genes. The extent to which MZ twins are more alike than DZ twins is therefore assumed to reflect additional genetic sharing. Heritability is defined as the proportion of the total phenotypic variance that is attributable to genetic variance ^1S and is calculated by means of structural equation modeling. Structural equation modeling quantifies sources of individual variation by decomposing the observed phenotypic variance into genetic and environmental variance. The genetic contribution can be further divided into an additive (A) genetic variance component (representing the influence of alleles at multiple loci acting in an additive manner) and a non-additive (D) genetic variance component (representing intra-locus interaction (dominance) and inter-locus interaction (epistasis) of alleles). The environmental component can be subdivided into a common (C) environmental variance component (representing environmental factors affecting both twins in a pair, a source of similarity) and random (E) environmental variance component

(representing environmental factors not shared by twins, a source of dissimilarity that includes random factors and measurement errors) ¹⁶.

To evaluate the relative importance of genetic and environmental influences on the number of hard drusen, structural equation modeiing was used to fit different models to the observed data under a number of standard assumptions, i.e. no gene-environment interaction, random mating and equal iπtra-pairwise environment in MZ and DZ twin pairs. Stippled fundus was considered a separate phenotype, as opposed to the quasi-continuum found in the rest of the population; hence five subjects with stippled fundi and their twins (8 subjects altogether) were excluded from the heritability analysis. Thus remained 116 monozygotic and 96 dizygotic twins. To deal with the highly discrete, skewed and non-normal distribution of the total number of drusen, a liability-threshold model was used ¹⁷. For this purpose the number of drusen per subject was grouped into the following categories: <1 , 1-5, 6-10, 11-20, 21-40, >40. The liability-threshold model is based on the notion of an underlying bivariate normal liability variable which is related to the category of the number of drusen by means of a set of thresholds, e.g. if the liability of a twin is between the 1^st and 2^nd threshold, the total number of drusen for this individual is between 1 and 5. The liability variable is then decomposed into genetic and environmental components using methods of quantitative genetics. The respective proportions of variance (including heritability of liability) are estimated using the maximum likelihood method, as implemented in the MX software package ^1S. To adjust for the effects of age and sex, a linear relationship between the mean liability and the 2 covariates was assumed, resulting in two additional regression coefficients to be estimated.

Casewise concordance describes the risk of having a certain phenotype given that the twin partner has this phenotype and is calculated from the formula 2C/ (2C+D) where C is the number of concordant pairs and D is the number of discordant pairs. A confidence interval for the observed value can be calculated as previously described ¹⁹.

The traditional Mest used for comparison of means in a two-sample case is based on the assumptions of normality and independent observations. However, not ai! variables in the present dataset are normally distributed. Moreover, there is potential interdependence between the observations, since they consist of members of twin pairs (i.e. the so-called "clustering"), To deal with this problem we have applied a linear regression analysis utilizing the so-called robust covariance matrix estimation which takes clustering into account ^2α;Z1 as implemented in the statistical package Stata 9 ²² Thus, the reported p-values of Table 2 regarding the comparison of MZ and DZ twins are robust with respect to deviations from normality and presence of intrapair correlation.

Results: Examination of the study population of 58 monozygotic and 52 dizygotic same-sex twin pairs of both sexes aged 20-46 years demonstrated that the monozygotic and dizygotic twin groups were comparable with regard to age, sex distribution, drusen number, smoking and other study variables, except that the dizygotic twins had slightly higher plasma lipid concentrations (Table 2). Epiretinal fibrosis was found unilaterally in two subjects, a nevus with overlying drusen unilaterally in two subjects, scarring from multifocal choroiditis bilaterally in one subject, and a single drusen of diameter >63μ - <125μ unilaterally in two subjects. All these findings were made in only one member of a twin pair. The presence of one or more small hard drusen in at least one eye was detected in 212 (96%) of out 220 subjects (Fig. 3). The mean number of smalt hard drusen was 6.0 in MZ twins and 8.5 in DZ twins (p = 0.22) (Table 2). A masked recounting of 22 random subjects demonstrated a weighted kappa statistic of 0.67 indicating substantial agreement in respect to the categories of drusen number used in the threshold-liability model. Five or more drusen per eye (the mean of a subject's two eyes) were found in 89 out of 220 subjects (40%). Five subjects (2.2%) demonstrated a stippled, granular fundus appearance in both eyes, with innumerable drusen, several hundreds or thousands per eye. In the remainder of the population, all subjects had less than 200 drusen per eye. The five subjects with stippled fundi were from 4 dizygotic female twin pairs. Their systemic characteristics were comparable to the remaining study population, data not shown.

In the 89 subjects with more than 5 drusen per eye, the distribution of subjects by drusen pattern was: scattered drusen 66 subjects, macular drusen 18 subjects, and stippled fundus 5 subjects (Table 3). In the 26 subjects with more than 20 drusen per eye 16 had predominately macular drusen, 5 had scattered drusen and 5 presented the stippled fundus phenotype. The pattern was the same in both eyes of all subjects.

Structural equation modeling yielded the superior fit using an AE model, regardless of whether the modeling was started using an ADE or an ACE model (Table 4). Age was the only covariate that reached statistical significance, the number of drusen increasing with age (Fig. 4). The estimated effects of age and sex (0 for males, 1 for females) on mean liability were -0.04 (CI_S5 -0.07,-0.02) and -0.34 (Cl₉₅ -0.69, 0.01 ), respectively. No statistically significant association was found between drusen number and sex, smoking, serum lipids, fasting blood glucose, or blood pressure. After adjustment for age and sex, the heritability of the number of small hard drusen was 63 (Cl₉₅ 43-77) % and the effect of random, non-shared environment was 37 (Cl₉₅ 23-57) %. This proportion includes the effect of random errors of measurement. Casewise concordance was higher in MZ twins than in DZ twins in the categories scattered drusen and macular drusen. Stippling of the fundus was not seen among MZ twins. For the phenotype ≥20 drusen the casewise concordance was 0.86 (Ci₉₅ 0.33,1.0) in MZ twins and 0.29 (Cl₉₅ 0.04,0.65) in DZ twins (Table 3). When applying structural equation modeling on the data with a threshold of >20 drusen per eye the best fit calculated by the use of the Akaike Information Criterion (AIC) was a DE model with a heritability of 99 % (Cl₉₅85%-100%) if the pairs with stippled fundus type were included and 99 % (CI₉₅82%-100%) if the pairs with stippled fundus type were excluded. In a DCE model the heritability of liability came out as 91 % (Cl₉₅ 36%- 100%), and 88% (CI₉₅29%-100%) respectively.

Comment: In elderly people, large numbers of hard drusen have previously been shown to predict the development of soft drusen ^3;4, but the significance of small hard drusen in younger subjects with normal visual function remains unknown ²³. Drusen do not appear to have a fundamentally different composition dependent on whether they are clearly circumscribed (hard drusen) or diffusely circumscribed (soft drusen) ²⁴.

Small drusen are not associated with specific histological characteristics of the drusen, but they tend to be associated with deflection of overlying photoreceptors that are otherwise of normal appearance, whereas larger drusen are associated with photoreceptor degeneration ²⁵. One or more drusen of any size within the macula in at least one eye has previously been found in 95.5% of a population of 4926 individuals between the ages of 43 to 86 years ². We found at least one small hard drusen in 96% of individuals aged 20-46 y. The drusen counted in this study have features in common with the Wisconsin AMD grading system categories hard distinct and hard indistinct drusen ¹⁴. Nevertheless, a number of fundamental differences exist between our methods of fundus photography and fundus grading and the Wisconsin AMD grading ¹⁴ and the Internationa! Classification and Grading System of ARM and AMD ^2S. (a) we studied subjects under the age of 50 , whereas subject younger than 50 y of age are excluded, by convention, from the aforementioned AMD protocols, (b) age-related maculopathy is not conventionally diagnosed unless soft drusen >63 μm or more advanced small hard drusen areas are present; again causing all but two of our participants to be excluded, (c) we did fundus photography in red-free illumination, which depicts smail hard drusen at considerably higher contrast than the color diapositϊve fundus photography techniques used in previous studies, and our use of digital photography provided immediate feed-back control of image focus and luminosity, enabling interactive adjustment of exposure settings, (d) the grader was allowed to use digital adjustment of the image so as to facilitate the identification of drusen, (e) we used a total small hard drusen area count rather than a subjective summation of total drusen area to quantify the amount of drusen, this approach being facilitated by the small hard drusen seen in the present study population being of apparently uniform size, whereas ARM and AMD often presents with drusen of widely differing dimensions, (f) most of our subjects had little evidence of a predominantly macular location of their small hard drusen, only 18 subjects demonstrating a distinct macular drusen pattern whereas 66 subjects showing the scattered drusen pattern among subjects with 5 or more drusen per eye (Table 3). When analyzed as a continuous trait the heritabiiity of smail hard drusen was 63% (Cl₉₅43% -77%). When analyzed as a dichotomous trait, 20 or more drusen per eye versus less than 20 drusen the heritabiiity was as high as 99% (CIg₅82%- 100%). The lower heritabiiity achieved when including the few, scattered drusen could be caused by the fact that the error of measurement is more critical in the low drusen number, but it is plausible that it is a high number of drusen that is the heritable phenotype.

The present study suggests that the presence, number, and, potentially, the distribution in the fundus of the eye of small hard drusen number in subjects who are in otherwise good ocular health may serve to predict who is going to develop ARM/AMD, to identify subjects where genotyping for ARM/AMD-related genes is warranted, to monitor progression of a pre-ARM/pre-AMD phenotype, and to monitor the effect of interventions aimed at reducing the risk of developing ARM/AMD and loosing sight from ARM/AMD. In the present study of small hard drusen, we were able to demonstrate an increasing number of small hard drusen with increasing age. This was facilitated by small hard drusen areas compatible with AMD being nearly completely absent in the study population, the population presumably being young enough for small hard drusen not to have begun to be replaced by other fundus small hard drusen area types. In the elderly, the prevalence of small hard drusen has been shown to decrease with age in concert with an increasing prevalence of larger drusen². Large number of small hard drusen (more than 8) has been found to be associated with an increase in the incidence of soft drusen and pigment abnormalities³ suggesting that larger drusen may develop from small drusen.

Recent studies have implicated genes of the complement system and other immunological factors in the pathogenesis of AMD ^31~34, as well as genes of unknown function ^35i3e. Our study shows that having more than 20 drusen per eye in young adulthood is highly hereditary. Previous studies have shown that in older subjects this feature is associated with AMD^3;4.

Table 2. Clinical characteristics of healthy monozygotic and dizygotic twins*

Numbers in mean (SD) except pairs, sex, and numbers of smokers in actual numbers.

* After exclusion of pairs in which one or both subjects demonstrated the stippled fundus phenotype. fRobust t-test (adjusted for clustering) φ capillary blood samples Tabie 3. Classification of small hard drυsen in healthy twins with 5 or more drusen per eye (n=89)

* including stippled fundus phenotype t exciuding stippled fundus phenotype

Table 4. Distribution modeling of small hard drusen in healthy twins

Boldface type indicates best fitting model for liability to develop a total number of drusen per subject in a given range (categories: <1, 1-5, 6-10, 11-20, 21-40, >40) after correction for the effect of age and sex. Twin pairs with individuals exhibiting stippled fundus were excluded from the analysis. Model components: A, additive genetic factors; C, shared environment; D, non-addittve genetic factors; E, unshared environment. | Proportion of total variation attributable to model component, 95% confidence interval in parentheses. -2InL minus 2 times log-likelihood of the data; AIC, Akaike's information criterion and p-value corresponding to the likelihood ratio test versus the ACE model. Both the ACE and the ADE model were simplifiable to an AE model.

References

1. Bressler NM₁ Bressler SB, West SK₁ et al. The grading and prevalence of macular degeneration in Chesapeake Bay watermen. Arch Ophthalmol

1989; 107:847-52.

2. Klein R, Klein BE, Linton KL. Prevalence of age-related macuiopathy. The Beaver Dam Eye Study. Ophthalmology 1992;99:933-43.

3. Klein R, Klein BE, Tomany SC, et al. Ten-year incidence and progression of age-related macuiopathy: The Beaver Dam eye study. Ophthalmology

2002; 109: 1767-79.

4. Bressler NM, Munoz B, Maguire MG, et al. Five-year incidence and disappearance of drusen and retinal pigment epithelial abnormalities. Waterman study. Arch Ophthalmol 1995;113:301-8.

5. Seddon JM₁ Cote J₁ Page WF₁ et al. The US twin study of age-related macular degeneration: relative roles of genetic and environmental influences. Arch Ophthalmol 2005; 123:321 -7.

6. Hammond CJ₁ Webster AR₁ Snieder H₁ et al. Genetic influence on early age- related macuiopathy: a twin study. Ophthalmology 2002; 109:730-6.

7. Kyvik KO, Christensen K, Skytthe A₁ et al. The Danish Twin Register. Dan Med

Bull 1996;43:467-70.

8. Scheurlen W₁ von Stockhauseπ HB, Kreth HW. Diagnose der Monozygotie bei Mehriingeπ durch DNA-Fingerprinting. Monatsschr Kinderheflkd 1991 ;139:30-2.

9. Schousboe K, Visscher PM, Henriksen JE, et al. Twin study of genetic and environmental influences on glucose tolerance and indices of insulin sensitivity and secretion. Diabetologia 2003;46:1276-83.

10. Kessel L, Hougaard JL, Sander B₁ et al. Lens ageing as an indicator of tissue damage associated with smoking and non-enzymatic g!ycation~a twin study. Diabetologia 2002:45:1457-62. 11. Hoυgaard JL, Kessel L, Sander B, et al. Evaluation of heredity as a determinant of retinal nerve fiber layer thickness as measured by optical coherence tomography. Invest Ophthalmol Vis Set 2003;44:3011-6.

12. Taarnhoj NC₁ Munch IC, Kyvik KO, et al. Heritability of cilioretinal arteries: a twin study. Invest Ophthaimol Vis Sci 2005;46:3850-4.

13. Wang M, Sander B, Ia Cour M, Larsen M. Clinical characteristics of subretinal deposits in central serous chorioretinopathy. Acta Ophthaimol Scand 2005;83:691-6.

14. Klein R, Davis MD, Magli YL, et al. The Wisconsin age-related maculopathy grading system. Ophthalmology 1991 ;98: 1128-34.

15. Day INM, Humphries SE. Genetics of Common Diseases. Oxford, UK: BIOS Scientific Publishers Ltd, 1997; 19-32.

16. Plomin R, DeFries JC, McCleam G.E., Rutter M. Behavioral Genetics., Third ed. New York, NY 10010: W.H. Freeman and Company, 1997;305-10.

17. Neale MC, Cardon LR. Methodology for genetic studies of twins and families.

Dordrecht, the Netherlands: Klϋwer Academic Publishers, 1992.

18. Neale MC BSXGMH. MX Manual, MX Statistical Modeling. 6. Richmond; 2003.

2003. Ref Type: Computer Program

19. Witte JS, Carlin JB, Hopper JL. Likelihood-based approach to estimating twin concordance for dichotomous traits. Genet Epidemiol 1999; 16:290-304.

20. Rogers W: H. Regression standard error in clustered samples. Stata Technical Bulletin 1993;13:19-23.

21. Williams RL. A note on robust variance estimation for cluster-correlated data. Biometrics 2000;56:645-6.

22. StataCorp. Stata Statistica! Software. College Station, TXD. 2005. Ref Type: Computer Program 23. Sarks SH, Arnold JJ, Kiliingsworth MC, Sarks JP. Early drusen formation in the normal and aging eye and their relation to age related maculopathy: a ciinicopathological study. Br J Ophthalmol 1999;83:358-68.

24. Hageman GS, Mullins RF. Molecular composition of drusen as related to substructural phenotype. MoI Vis 1999;5:28.

25. Johnson PT, Lewis GP, Taiaga KC₁ et al. Drusen-associated degeneration in the retina. Invest Ophthalmol Vis Sci 2003;44:4481-8.

26. Bird AC₁ Bressier NM, Bressler SB, et al. An international classification and grading system for age-related maculopathy and age-related macular degeneration. The Internationa! ARM Epidemiological Study Group. Surv

OphthalmoM 995:39:367-74.

27. Fraser-Bell S, Wu J, Klein R, et al. Smoking, alcohol intake, estrogen use, and age-related macular degeneration in Latinos: the Los Angeles Latino Eye Study. Am J Ophthalmol 2006; 141.79-87.

28. Thornton J, Edwards R, Mitchell P, et al. Smoking and age-related macular degeneration: a review of association. Eye 2005;19:935-44.

29. Khan JC, Thurlby DA, Shahid H, et al. Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisatioπ. Br J Ophthalmol 2006;90:75-80.

30. van Leeuwen R, lkram MK, Vingerling JR, et al. Blood pressure, atherosclerosis, and the incidence of age-related maculopathy: the Rotterdam Study, invest Ophthalmol Vis Sci 2003;44:3771-7.

31. Edwards AO, Ritter R, III, Abel KJ, et al. Complement factor H polymorphism and age-related macular degeneration. Science 2005; 308: 421-4.

32. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 2005; 308:419- 21. 33. Klein RJ, Zeiss C, Chew EY₁ et a!. Complement factor H polymorphism in age- related macular degeneration. Science 2005;308:385-9.

34. Gold B, Merriam JE, Zernant J, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 2006;38:458-62.

35. Schmidt S, Hauser MA, Scott WK, et al. Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration. Am J Hum Genet 2006;78:852-64.

36. Rivera A, Fisher SA, Fritsche LG, et al. Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum MoI Genet 2005; 14:3227-36.

Claims

1. A method for determining a predisposition for acquiring macula degeneration in an individual, comprising

determining the number and/or position of said small hard drusen area(s),

2. The method according to claim 1 , wherein at least two images of different parts of retina are used for detecting small hard drusen area(s) in one eye, such as at least four images,

3. The method according to claim 1 or 2, wherein the predetermined distance to macula corresponds to the distance between macula and the optic nerve or iess, such as a distance corresponding to Vz the distance between macula and the optic nerve head or less, such as a distance corresponding to 1/4 the distance between macula and the optic nerve head or less.

4. The method according to any of the preceding claims, wherein a number of at least 8 smal! hard drusen is indicative of a predisposition for acquiring macula degeneration at a later stage in life.

The method according to any of the preceding claims, wherein the individual is between 20 and 46 years old.

5. The method according to any of the preceding claims, wherein the image is presented on a medium selected from dias, paper photos or digital photos.

6. The method according to any of the preceding claims, wherein the image is a colour image.

7. The method according to any of the preceding claims, wherein the small hard drusen are identified as extrema of the image.

8. The method according to any of the preceding claims, wherein the extrema are identified by filtering the image.

9. The method according to any of the preceding claims, wherein the extrema are corrected with respect to the background variation.

11. The method according to any of the preceding claims, further comprising detection of a region comprising the optic nerve head.

12. The method according to any of the preceding ciaims, further comprising detection of a region comprising the macula

13. The method according to any of the preceding claims, further comprising determination of at least one polymorphism is determined in a gene selected from genes of the complement system or in a chromosome region containing said gene.

14. The method according to claim 13, wherein the at least one polymorphism is determined in a gene selected from complement factor H gene, LOC387715 gene, factor B gene and/or complement component 2 gene or chromosome regions containing said genes.

15. The method according to claims 13 to 4, wherein at least one of the polymorphisms is determined in a non-coding region of a gene such as an intron or in a region controlling expression of the gene.

16. The method of claim 15, wherein the at least one of the polymorphisms is determined in a non-coding region of the complement factor H gene, LOC387715 gene, factor B gene and/pr complement component 2 gene such as an intron region.

17. The method of claim 15, wherein the at least one polymorphism is determined in the region comprising a nucleotide sequence controliing expression of complement factor H gene, LOC387715 gene, factor B gene and/or complement component 2 gene, such as a promotor region.

18. A method for classifying a fundus image comprising

- assessing the presence or absence of at least one small hard drusen area(s) by the method as defined in any of the claims 1-12,

classifying the fundus image into at least two classes, wherein above 6 small hard drusen areas in at least one eye, and/or a position of smali hard drusen within a predetermined distance to the macula leads to classification of said image as a an image of an individual at risk for acquiring macula degeneration at a later stage in life.

19. A system for determining a predisposition for acquiring macula degeneration in an individual, comprising

an algorithm for detecting, in at least one image of retina of said individual, small hard drusen area(s), and

20. The system according to claim 19, comprising means for carrying out one or more of the steps according to any of claims 1-17.

21. The system according to claim 19 or 20, comprising means for entering further information of said individual,

22. The system according to claim 21 , wherein the information is result of a genotype test as defined by any of the claims 13-17.

23. A system for classifying a fundus image comprising

- an algorithm for assessing the presence or absence of at least one small hard drusen area(s) by the method as defined in claim 18,

24. A method for monitoring an individual with respect to macula degeneration, said method comprising

detecting in at least one retina image from said individual small hard drusen area{s), and

determining the number and/or position of said small hard drusen area(s),

25. A computer readable medium comprising instructions for carrying out the method according to any of the preceding claims 1-18 or 24.

26. A software program comprising instructions for carrying out the method according to any of the preceding claims 1 -18 or 24.