WO2008110824A2

WO2008110824A2 - Diagnostic test for age-related macular degeneration (amd)

Info

Publication number: WO2008110824A2
Application number: PCT/GB2008/000917
Authority: WO
Inventors: Igor Bendik; Ozgür SANCAK; Wolfgang Schalch; Jonas Wittwer
Original assignee: Dsm Ip Assets B.V.; Wright, Simon, Mark
Priority date: 2007-03-14
Filing date: 2008-03-14
Publication date: 2008-09-18
Also published as: WO2008110283A2; WO2008110824A3

Abstract

A method of prevention of age-related macular degeneration (AMD) is provided where a sample is taken from an individual and the presence of a polymorphism associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or an SNP as shown in Table 1 of the specification, is detected. The individual can then be provided with a substance that can prevent or treat AMD, or can mitigate or alleviate symptoms of AMD. The substance is a medication comprising lutein and/or zeaxanthin and/or certain antioxidants (or a mixture thereof) is tailored to that individual.

Description

DIAGNOSTIC TEST FOR AGE-RELATED MACULAR DEGENERATION (AMD)

Field of the invention

The present invention relates to a novel diagnostic test for age-related macular degeneration and, subsequently, a method for the (treatment and/or) prevention of age related macular degeneration (AMD). It thus relates to the diagnosis and/or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in an individual (or subject), usually by determining susceptibility of the individual to age- related macular degeneration and, on the basis of that determination, selecting or identifying (and administering) a substance to the individual.

Background of the invention

As the most common cause of vision loss among people over the age of 60, macular degeneration impacts millions of older adults every year. The disease affects central vision and can sometimes make it difficult to read, drive or perform other activities requiring fine, detailed vision. When the macula is damaged, the eye loses its ability to see detail, such as small print, facial features or small objects. The damaged parts of the macula often cause scotomas, or localized areas of vision loss.

There are two types of the disease: dry macular degeneration and wet macular degeneration. Ninety percent of people who have macular degeneration have the dry form of the condition. In dry macular degeneration or atrophic macular degeneration, waste products may accumulate in the tissues underneath the macula forming yellowish deposits called drusen. The continued presence of drusen interferes with the blood flow to the retina and, in particular, to the macula. Less blood flow reduces the nourishment to the macula causing its light sensitive cells to stop working efficiently, or atrophy.

With wet macular degeneration, new weak blood vessels may grow in or under the retina causing fluid and blood to leak into the space under the macula. As a result, wet macular degeneration is sometimes called exudative macular degeneration, or described as choroidal neovascularization. The choroid is the area of blood vessels beneath the retina, and neovascularization refers to growth of new blood vessels in tissue. In choroidal neovascularization, blood vessels from the choroid grow into the macula. The most common early sign of dry macular degeneration is blurred vision. As fewer cells in the macula are able to function, people will see details less clearly in front of them, such as faces or words in a book. If the loss of these light-sensing cells becomes great, people may see a small - but growing - blind spot in the middle of their vision. The classic early symptom of wet macular degeneration is that straight lines appear crooked. This occurs when fluid from the leaking blood vessels gathers and lifts the macula, distorting vision. A small blind spot may also appear in wet macular degeneration, resulting in loss of one's central vision.

Regular eye exams are necessary for early detection of macular degeneration since symptoms may or may not be present in people who have the disease. Early drusen can be seen in an eye exam before symptoms develop.

The disease typically develops over an extended period of time and becomes apparent mostly not before it has reached an advanced stage. Further, while the etiology so far has remained largely unclear it has been reported that susceptibility for AMD may inter alia be genetically predetermined.

The present invention relates to the identification of those subjects which may have increased risk for developing (dry and/or wet) AMD and with the treatment and, particularly, prevention of AMD in those subjects suitably at an early as possible point in time.

Summary of the invention

A robust genetic test for determining susceptibility to AMD (in humans) has now been developed. The present inventors have discovered single nucleotide polymorphisms (SNPs) that are associated with susceptibility to AMD. The identification of these polymorphisms provides the basis for a diagnostic test to identify individuals at risk of AMD or confirm diagnosis of the condition by screening for specific molecular markers. The predictive power of the diagnostic test can be magnified by using models that involve combining the results of typing one or more of the defined SNPs. Subjects that are determined to be susceptible to the disease may then follow preventative methodologies such as appropriate diets and treatments, in order to delay or prevent the onset of AMD, or to alleviate the symptoms in an individual that already has the condition. Accordingly, the invention provides a method of determining whether an individual is susceptible to or has AMD, the method comprising detecting a polymorphism (usually in a sample from the individual), or typing the nucleotide present in the genome of the individual for the presence of a polymorphism (or SNP), where the polymorphism is one or more of those identified in Table 1, and/or at one or more positions which are in linkage disequilibrium with any one of these positions. One can thereby determine whether the individual is susceptible to, or has, AMD.

The present invention also relates to a method for treatment and/or prevention of age- related macular degeneration (AMD), which method comprises: (a) detecting one or more polymorphism(s), usually in a sample taken from a subject (or individual, the terms are used interchangeably), wherein the polymorphism is as identified in Table 1 or is near, or is associated with (an enzyme which is a) dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment; and

(b) providing an effective amount of a substance to the individual, which substance can, or is able to, prevent or treat AMD or mitigate or alleviate symptoms of AMD.

Suitably the substance is a (preferably macular) carotenoid, in particular a xanthophyll, such as lutein and/or zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail, as described later).

The present invention also provides a method of determining a substance to be administered to an individual, which individual may be susceptible of having age-related macular degeneration AMD (which term includes a (wet or dry) age-related macular degeneration-related disorder or condition unless otherwise specified), the method comprising: a) detecting a polymorphism in a sample, which may indicate the susceptibility of the individual to age-related macular degeneration (AMD); and b) on the basis of the determination in (a), identifying or selecting a substance capable of preventing and/or treating age-related macular degeneration (AMD) in that individual.

The method may additionally comprise: c) providing (such as administering or communicating) the substance (or its identity) to the individual. The invention further provides: i) means capable of detecting the SNP (or allelic variant) related or associated with age- related macular degeneration in an individual and means for providing (such as administering or communicating) a substance (or its identity) capable of preventing or treating age-related macular degeneration to the individual; ii) a kit for carrying out the method of the invention comprising means for detecting a SNP or allelic variant or haplotype or combination of SNPs and an effective amount of the substance, e.g. a (preferably macular) carotenoid, in particular a xanthophyll, such as lutein and/or (optionally meso-) zeaxanthin and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail); iii) a method of preparing a customised or personalised composition for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:

(a) detecting a polymorphism in a sample, which may indicate whether the individual is susceptible to age-related macular degeneration, or age-related macular degeneration-related disorder by a method of the invention; and

(b) preparing a composition suitable for, or tailored to, the individual; iv) a method of providing a customised composition, comprising providing a composition suitable for a subject which is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the individual has been (eg. genetically) determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), using one or more of the polymorphisms disclosed herein; v) a method of identifying a substance for the prevention (which includes delaying progression or preventing occurrence of) or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:

(a) contacting an allelic variant polypeptide related to or associated with AMD or a polynucleotide which encodes an allelic variant related to or associated with AMD with a test agent; and (b) determining whether the substance is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide, and providing (such as administering or communicating) the substance (or its identity) to an individual; vi) use of a compound which is therapeutic for age-related macular degeneration (or a age-related macular degeneration-related disorder), optionally in the manufacture of a medicament, for the prevention or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in a individual that has been identified as being susceptible to age-related macular degeneration by a method of the invention; vii) a method of treating an individual for age-related macular degeneration (or an age- related macular degeneration-related disorder), the method comprising administering to the individual an (effective amount of a) therapeutic substance or compound which can prevent or treat AMD or a related disorder, wherein the individual has been identified as being susceptible to age-related macular degeneration, or an age-related macular degeneration-related disorder, by a method of the invention; viii) a database comprising information relating to age-related macular degeneration allelic variants and optionally their association with age-related macular degeneration related disorder(s) and/or substances capable of preventing or treating age-related macular degeneration; ix) a method for determining whether an individual is susceptible to age-related macular degeneration, or an age-related macular degeneration-related disorder, the method comprising:

(a) inputting data of one or more haplotypes or allelic variant(s) (or combinations of SNPs or haplotypes) present in the subject to a computer system;

(b) comparing the data to a computer database, which database comprises information relating to allelic variants related or associated with AMD and the age-related macular degeneration related disorder susceptibility associated with the variants; and

(c) determining on the basis of the comparison whether the individual is susceptible to age-related macular degeneration or a related disorder; x) a computer program comprising program code means that, when executed on a computer system, instructs the computer system to perform a method according to the invention; xi) a computer program product comprising a computer-readable storage medium having recorded thereon a computer program according to the invention; xii) a computer program product comprising program code means, on a carrier wave or stored on a computer readable medium, which program code means, when executed on a computer system, instructs the computer system to perform a method according to the invention; xiii) a computer system arranged to perform a method according to the invention, such as comprising: (a) means for receiving data of the one or more age-related macular degeneration allelic variant(s) present in the individual;

(b) a module for comparing the data with a database comprising information relating to age-related macular degeneration allelic variants and the age-related macular degeneration related disorder susceptibility associated with the variants; and (c) means for determining, on the basis of said comparison whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder); xiv) a method of preparing a customised composition for an individual which is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder), the method comprising:

(a) determining whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder) by a method of the invention;

(b) determining (such as electronically generating) a customised composition suitable for the individual;

(c) optionally, generating (e.g. electronic) manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition formulation; and

(d) manufacturing the customised food (according to the manufacturing instructions); xiv) use of a computer system of the invention to make a customised composition product; or

(xv) a kit for carrying out the method of the invention comprising a probe or primer(s) that is capable of detecting a polymorphism related to AMD. Prevention (of AMD) in this specification includes assisting in preventing AMD, mitigating or alleviating one or more symptoms of AMD, and delaying the progression or preventing occurrence of AMD. In addition, AMD includes AMD-related disorders. The detection may thus confirm a diagnosis of AMD by screening for particular molecular markers.

Brief Description of the Sequences SEQ ID NOs: 1 to 70 show the polynucleotide sequences encompassing the SNPs used in the invention.

Detailed description of the invention

The present invention provides a method for determining whether or not an individual, is susceptible to, or has, AMD. In the context of the present invention, the term "susceptible" is taken to mean that the individual is likely to develop or has a predisposition to the development of AMD. An individual that is susceptible or predisposed to the condition may have a greater than 60% chance of demonstrating symptoms that are associated with the condition. Accordingly, an individual that is susceptible may have a greater than 70%, 80% or 90% chance of exhibiting symptoms of the condition at some stage in their life. For example, in a sample of 100 subjects that are diagnosed as susceptible, at least 60, at least 70, at least 80, or at least 90 of the subjects will display symptoms of the condition, hi a preferred embodiment, all subjects that are diagnosed as susceptible to AMD will display symptoms of the condition. An individual may be tested by a method of the invention at any age, for example from 30 to 80 years old, 40 to 70 years old, from 50 to 60 years old, from 40 to 60 years old, or from 50 to 70 years old. Preferably the individual is tested young, for example before they are 30 or 40. The individual can be tested before any symptoms of AMD are apparent. One aim is therefore to take preventative measures before the symptoms of AMD occur, such as modifying the diet, providing supplements and/or avoiding exposure to certain allergens, in order to delay or prevent the onset of or alleviate the symptoms of AMD. However, when the method is used to diagnose or confirm the diagnosis of the condition there is no preferable age of the individual. This will enable the individual, physician or optician to take an appropriate course of action for treatment of the condition or for alleviating the symptoms of the condition.

The present invention provides a method of determining whether an individual is susceptible to or has AMD, the method comprising determining the presence (or absence) in the genome of the individual, a polymorphism as identified in Table 1, and/or at one or more positions which are in linkage disequilibrium with any one of these positions, and thereby determining whether the individual is susceptible to or has AMD. Typically, the nucleotide present at the same location or position or locus on both homologous chromosomes will be determined. An individual may therefore be determined to be homozygous for a first allele, heterozygous or homozygous for a second allele of the SNP.

In a more preferred embodiment of the invention, the one or more positions (or polymorphisms) that are typed are: a) ABCA4 of Table 1. eg. rs6657239 or rsl 800549; b) BCDO2 of Table 1, eg. rs7123686 or rs2676377; c) CST3 of Table 1, eg. rsl 1504 or rsl 135147; d) FBLN5 of Table 1, eg. rs929608, rs2430347, rs 2474028 or rsl7127768; e) ILIA of Table 1, eg. rsl304037, rsl7561, rs2856841 or rs2856837; f) NFE2L2 of Table 1, eg. rs2364731, rs7568033, rs6433659, or rs3813259; g) RPE65 of Table 1, eg. rs3118416, rs2182315, rs3118418 or rsl2145904; h) SODl of Table 1, eg. rs3216079; i) TF of Table 2, eg. rsl 130459, rsl2769, rsl800669 or rs8649. Or one or more positions which are in linkage disequilibrium with any one of these positions.

Any one of the polymorphisms defined herein may be typed directly, in other words by determining the nucleotide present at that position, or indirectly, for example by determining the nucleotide present at another polymorphic position that is in linkage disequilibrium with said polymorphic position. Examples of SNPs that are in linkage disequilibrium with the SNPs of the invention and can therefore be used to determine susceptibility to AMD are identified later. Polymorphisms which are in linkage disequilibrium with each other in a population are typically found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40%, at least 50%, at least 70% or at least 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus a polymorphism which is not a functional susceptibility polymorphism, but is in linkage disequilibrium with a functional polymorphism, may act as a marker indicating the presence of the functional polymorphism.

Polymorphisms which are in linkage disequilibrium with the polymorphisms mentioned herein are typically located within 9mb, preferably within 5mb, within 2mb, within lmb, within 500kb, within 400kb, within 200kb, within 100kb, within 50kb, within 10kb, within 5kb, within lkb, within 500bp, within lOObp, within 50bp or within lObp of the polymorphism. Any number and any combination of the SNP positions as described herein may be typed to carry out the invention. Preferably at least 2 SNP positions are typed, more preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 45 SNP positions are typed. The number of SNP positions typed may be from 1 to 50, from 2 to 40, from 5 to 30 or from 5 to 20. hi a more preferred embodiment, the SNP positions are selected from those: a) ABCA4 of Table 1. eg. rs6657239 or rsl 800549; b) BCDO2 of Table 1, eg. rs7123686 or rs2676377; c) RPE65 of Table 1, eg. rs3118416, rs2182315, rs3118418 or rsl 2145904; d) SODl of Table 1, eg. rs3216079. Accordingly, any of these SNPs or any SNPs that are in linkage disequilibrium with any of these SNPs may be typed. Suitably at least 2 of these preferred SNPs or SNPs in linkage disequilibrium are typed. More preferably at least 3, 4, 5, 6, 7, 8 or all preferred positions (or polymorphisms) are typed.

Typing the nucleotide present in the genome at a position equivalent to that identified in Table 1 may mean that the nucleotide present at this position in a sequence corresponding exactly with the sequence identified in Table 1 is typed. However, it will be understood that the exact sequences presented in Table 1 will not necessarily be present in the individual to be tested. Typing the nucleotide present may therefore be at the position identified in Table 1 or at an equivalent or corresponding position in the sequence. The term equivalent as used herein therefore means at or at a position corresponding to position identified. The sequence and thus the position of the SNP could for example vary because of deletions or additions of nucleotides in the individual's genome. Those skilled in the art will be able to determine a position that corresponds to or is equivalent to the polymorphism, using for example a computer program such as GAP, BESTFIT, COMPARE, ALIGN, PILEUP or BLAST. The UWGCG Package provides programs including GAP, BESTFIT, COMPARE, ALIGN and PILEUP that can be used to calculate homology or line up sequences (for example used on their default settings). The BLAST algorithm can also be used to compare or line up two sequences, typically on its default settings. Software for performing a BLAST comparison of two sequences is pubicly available through the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.govΛ. This algorithm is further described below. Similar publicly available tools for the alignment and comparison of sequences may be found on the European Bioinformatics Institute website fhttp://www.ebi.ac.uk), for example the ALIGN and CLUSTALW programs.

The inventors have further discovered models for using the SNPs of the invention to determine whether or not an individual is susceptible to or has AMD. It will be appreciated that many different models with varying degrees of predictive power, are possible using any number or combination of the SNPs of the invention.

Models for use in predicting susceptibility to AMD are illustrated below, and utilize the SNPs from Table 1. These may be performed either singly or in combination to provide a prediction of susceptibility or diagnosis of AMD. Each is designed to favour the determination of AMD cases over non-AMD cases. The effect is to minimise the identification of false negatives and to wrongly predict an individual is susceptible to AMD.

The models of the invention are now described in more detail. Each of the following 5 models may be conducted on their own for the determination of susceptibility to AMD. Alternatively, the models may be combined such that the result of each model acts as a vote towards the final result. For each model, each SNP is genotyped by determining the nucleotide present on both homologous chromosomes.

In a first model, a subject is classified as being susceptible to AMD according to the table below. If none of the 2 genotypes apply, the individual is classified as susceptible to AMD.

The individual is classified as susceptible to AMD if they carry the risk alleles with AMD for rs7123686 or rs2676377. Alternatively, the individual can be classified as having AMD by the presence of the following SNPs.

Determination of susceptibility

The individual may be susceptible to age-related macular degeneration, or an age- related macular degeneration -related disorder. This may mean that they are at risk of, or have a predisposition to, age-related macular degeneration or an age-related macular degeneration-related disorder. That individual may, or may not, in fact, necessarily have an increased risk or susceptibility, depending on the determination. The determination may find that the person has an increased risk, or (on the contrary) a decreased risk, of said disorder.

The detection or typing in (a) may thus comprise typing the nucleotide (present in the genome of the individual) at a position equivalent to position 26 in one or more of the sequences identified in the attached sequence listing (eg. any of SEQ ID NOs: 1 to 70).

Stage (a) may thus additionally comprise preparing or obtaining a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or nutritional profile (or identity) of the individual. This may assist in determining the susceptibility (such as risk or predisposition) to a disorder. Thus, the determination may comprise: i) conducting or performing a genome or genetic analysis of the individual; and ii) preparing a pharmacogenomic, nutrigenomic, nutrigenetic, transcriptomic, epigenetic or proteomic and/or metabolomic profile and/or identity based on personal and/or clinical information from or about the individual; iii) optionally, performing a test or assay (such as on a biological sample from the individual) that can indicate susceptibility (to age-related macular degeneration).

In (i), this may involve determining an individual's genotype (for the respective loci). It can comprise identifying an allelic variant, a polymorphism (such as an SNP) a haplotype or combination thereof (e.g. SNP and haplotype) or genetic predisposition (to the relevant disorder). One may therefore be able to draw up a genetic profile of the individual, preferably relevant to the disorder.

The determination may, alternatively or in addition to (ii), comprise obtaining relevant information from, or about, that individual. That information may be personal and/or clinical information. The information may relate, directly or indirectly, to age- related macular degeneration (or an age-related macular degeneration-related disorder). Such information may comprise information concerning lifestyle, health, nutritional status, diet. Other personal information of relevance can include age, sex, weight and/or ethnic background. Clinical information may comprise current drug and/or vitamin regimes, current or past treatments, familial data, health risks, family background, medical conditions and/or allergies. It may therefore involve obtaining a patient's medical history or current health status and determining their nutritional profile. The individual may be able to provide this information, for example, by completing a questionnaire.

Detection of polymorphisms

The detection of polymorphisms according to the invention may comprise contacting a polynucleotide or protein of the individual with a specific binding agent for a polymorphism and determining whether the agent binds to the polynucleotide or protein, wherein binding of the agent can indicate the presence of the polymorphism, and a lack of binding of the agent can indicate the absence of the polymorphism.

The method is generally carried out in vitro on a sample from the individual, where the sample contains DNA from the individual. The sample typically comprises a body fluid and/or cells and may, for example, be obtained using a swab, such as a mouth or buccal swab. The sample may be a blood, urine, saliva, skin, buccal mucosa eg. (cheek) cell or hair root sample. The sample is typically processed before the method is carried out, for example DNA extraction may be carried out. The polynucleotide or protein in the sample may be cleaved either physically or chemically, for example using a suitable enzyme. In one embodiment the part of polynucleotide in the sample is copied or amplified, for example by cloning or using a PCR based method prior to detecting the polymorphism.

In the present invention, any one or more methods may comprise determining the presence or absence of one or more polymorphisms in the individual. The polymorphism is typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein of the individual. Such a polynucleotide is typically genomic DNA, mRNA or cDNA. The polymorphism may be detected by any suitable method such as those mentioned below. A specific binding agent is usually an agent that binds with preferential or high affinity to the protein or polypeptide having the polymorphism but does not bind or binds with only low affinity to other polypeptides or proteins. The specific binding agent may be a probe or primer(s). The probe may be a protein (such as an antibody) or an oligonucleotide. The probe may be labelled or may be capable of being labelled indirectly. The binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein. Generally, a polymorphism can be detected by determining the binding of the agent to the polymorphic polynucleotide or protein of the individual. However in one embodiment the agent is also able to bind the corresponding wild-type sequence, for example by binding the nucleotides or amino acids which flank the variant position, although the manner of binding to the wild-type sequence will be detectably different to the binding of a polynucleotide or protein containing the polymorphism.

The method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide that contains the polymorphism, allowing after binding the two probes to be ligated together by an appropriate ligase enzyme. However the presence of a single mismatch within one of the probes may disrupt binding and ligation. Thus ligated probes will only occur with a polynucleotide that contains the polymorphism, and therefore the detection of the ligated product may be used to determine the presence of the polymorphism.

In one embodiment the probe is used in a heteroduplex analysis based system. In such a system when the probe is bound to polynucleotide sequence containing the polymorphism it forms a heteroduplex at the site where the polymorphism occurs and hence does not form a double strand structure. Such a heteroduplex structure can be detected by the use of a single or double strand specific enzyme. Typically the probe is an RNA probe, the heteroduplex region is cleaved using RNAase H and the polymorphism is detected by detecting the cleavage products. The method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. Sci. 85, 4397-4401 (1998).

In one embodiment a PCR primer is used that primes a PCR reaction only if it binds a polynucleotide containing the polymorphism, for example a sequence-specific PCR system, and the presence of the polymorphism may be determined by detecting the PCR product. Preferably the region of the primer that is complementary to the polymorphism is at or near the 3' end of the primer. The presence of the polymorphism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system. In a preferred embodiment, one or more of the probes and/or primers are used in a Taqman assay to detect an allelic variant.

The specific binding agent may be capable of specifically binding the amino acid sequence encoded by a polymorphic sequence. For example, the agent may be an antibody or antibody fragment. The detection method may be based on an ELISA system. The method may be an RFLP based system. This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognised by a restriction enzyme. The presence of the polymorphism may be determined based on the change that the presence of the polymorphism makes to the mobility of the polynucleotide or protein during gel electrophoresis. In the case of a polynucleotide, single-stranded conformation analysis (SSCP) or denaturing gradient gel electrophoresis (DGGE) analysis may be used. In another method of detecting the polymorphism, a polynucleotide comprising the polymorphic region is sequenced across the region that contains the polymorphism to determine the presence of the polymorphism.

The presence of the polymorphism may be detected by means of fluorescence resonance energy transfer (FRET). In particular, the polymorphism may be detected by means of a dual hybridisation probe system. This method involves the use of two oligonucleotide probes that are located close to each other and that are complementary to an internal segment of a target polynucleotide of interest, where each of the two probes is labelled with a fluorophore. Any suitable fluorescent label or dye may be used as the fluorophore, such that the emission wavelength of the fluorophore on one probe (the donor) overlaps the excitation wavelength of the fluorophore on the second probe (the acceptor). A typical donor fluorophore is fluorescein (FAM), and typical acceptor fluorophores include Texas red, rhodamine, LC-640, LC-705 and cyanine 5 (Cy5).

In order for fluorescence resonance energy transfer to take place, the two fluorophores need to come into close proximity on hybridisation of both probes to the target. When the donor fluorophore is excited with an appropriate wavelength of light, the emission spectrum energy is transferred to the fluorophore on the acceptor probe resulting in its fluorescence. Therefore, detection of this wavelength of light, during excitation at the wavelength appropriate for the donor fluorophore, indicates hybridisation and close association of the fluorophores on the two probes. Each probe may be labelled with a fluorophore at one end such that the probe located upstream (5') is labelled at its 3' end, and the probe located downstream (3') is labelled at is 5' end. The gap between the two probes when bound to the target sequence may be from 1 to 20 nucleotides, preferably from 1 to 17 nucleotides, more preferably from 1 to 10 nucleotides, such as a gap of 1, 2, 4, 6, 8 or 10 nucleotides.

The first of the two probes may be designed to bind to a conserved sequence of the gene adjacent to a polymorphism and the second probe may be designed to bind to a region including one or more polymorphisms. Polymorphisms within the sequence of the gene targeted by the second probe can be detected by measuring the change in melting temperature caused by the resulting base mismatches. The extent of the change in the melting temperature will be dependent on the number and base types involved in the nucleotide polymorphisms.

Polymorphism typing may also be performed using a primer extension technique, hi this technique, the target region surrounding the polymorphic site is copied or amplified for example using PCR. A single base sequencing reaction is then performed using a primer that anneals one base away from the polymorphic site (allele-specific nucleotide incorporation). The primer extension product is then detected to determine the nucleotide present at the polymorphic site. There are several ways in which the extension product can be detected. In one detection method for example, fluorescently labelled dideoxynucleotide terminators are used to stop the extension reaction at the polymorphic site. Alternatively, mass-modified dideoxynucleotide terminators are used and the primer extension products are detected using mass spectrometry. By specifically labelling one or more of the terminators, the sequence of the extended primer, and hence the nucleotide present at the polymorphic site can be deduced. More than one reaction product can be analysed per reaction and consequently the nucleotide present on both homologous chromosomes can be determined if more than one terminator is specifically labelled.

Polynucleotides The invention further provides primers or probes that may be used in the detection of any of the SNPs defined herein for use in the determination of susceptibility to AMD. Polynucleotides of the invention may also be used as primers for primer extension reactions to detect the SNPs defined herein.

Such primers, probes and other polynucleotide fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of a full length polynucleotide sequence of the invention.

Suitable primer and primer extension sequences for use in the method of the invention can be designed from the genomic sequence around each SNP using the specific assay design functionality software in the Sequenom MassARRAY workstation package (Array design version 3.0 and Assay Editor version 3.0.4.6). Homologues of these polynucleotide sequences would also be suitable. Such homologues typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology, for example over a region of at least 15, 20, 30, 100 more contiguous nucleotides. The homology may be calculated on the basis of nucleotide identity (sometimes referred to as "hard homology").

For example the UWGCG Package provides the BESTFIT program that can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as default a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. ScL USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001. The homologous sequence typically differs by at least 1, 2, 5, 10, 20 or more mutations, which may be substitutions, deletions or insertions of nucleotides

The polynucleotides of the invention may be present in an isolated or substantially purified form. They may be mixed with carriers or diluents that will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of polynucleotides of the preparation.

Detector antibodies

A detector antibody is an antibody that is specific for one polymorphism but does not bind to any other polymorphism as described herein. Detector antibodies are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques. Antibodies may be raised against specific epitopes of the polypeptides of the invention. An antibody, or other compound, "specifically binds" to a polypeptide when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other polypeptides. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments that bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')₂ fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample (such as any such sample mentioned herein), which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising an antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, hereinafter the "immunogen". The fragment may be any of the fragments mentioned herein (typically at least 10 or at least 15 amino acids long). A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497). An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

Detection kit The invention also provides a kit that comprises means for typing one or more of the polymorphisms defined herein, or for determining the presence (or absence) of one or more AMD allelic variants or SNPs. hi particular, such means may include a specific binding agent, probe, primer pair or combination of primers, or antibody, including an antibody fragment, as defined herein which is capable of detecting or aiding detection of the polymorphisms defined herein. The primer pair or combination of primers may be sequence specific primers that only cause PCR amplification of a polynucleotide sequence comprising the polymorphism to be detected, as discussed herein. The primer or pair of primers may alternatively not be specific for the polymorphic nucleotide, but may be specific for the region upstream (5') and/or downstream (3'). These primers allow the region encompassing the polymorphic nucleotide to be copied. A kit suitable for use in the primer-extension technique may specifically include labelled dideoxynucleotide triphosphates (ddNTPs). These may for example be fluorescently labelled or mass modified to enable detection of the extension product and consequently determination of the nucleotide present at the polymorphic position. The kit may also comprise a specific binding agent, probe, primer, pair or combination of primers, or antibody that is capable of detecting the absence of the polymorphism. The kit may further comprise buffers or aqueous solutions. The kit may additionally comprise one or more other reagents or instruments that enable any of the embodiments of the method mentioned above to be carried out. Such reagents or instruments may include one or more of the following: a means to detect the binding of the agent to the polymorphism, a detectable label such as a fluorescent label, an enzyme able to act on a polynucleotide, typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide, suitable buffer(s) or aqueous solutions for enzyme reagents, PCR primers which bind to regions flanking the polymorphism as discussed herein, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from sample, a means to obtain a sample from the individual, such as swab or an instrument comprising a needle, or a support comprising wells on which detection reactions can be carried out. The kit may be, or include, an array such as a polynucleotide array comprising the specific binding agent, preferably a probe, of the invention. The kit typically includes a set of instructions for using the kit.

Treatments and further action based on SNP detection

According to a further aspect of the present invention, there is provided a method of determining a substance to be administered to an individual (or subject, the terms are used interchangeably), the method comprising: a) determining the susceptibility to AMD, for example by detecting a polymorphism (in a sample taken from an individual) wherein the polymorphism is as described above; and b) on the basis of the determination in a), identifying a substance capable of preventing and/or treating age-related macular degeneration in that individual.

The method may additionally comprise: c) providing (e.g. administering or communicating) the substance (or its identity) to the individual (or subject).

Step c) can thus comprise communicating the identity of that substance to the individual, for example proposing, suggesting or recommending that substance. For instance, this may involve supplementing a person's food or diet with said substance. Prevention (of AMD) in this specification can thus involve mitigating or alleviating one or more symptoms of AMD. Factors in the determination of susceptibility

The determination of susceptibility may also comprise preparing or obtaining a pharmacogenomic, nutritional profile (or identity) of the individual. This may assist in determining the susceptibility (such as risk or predisposition) to an AMD disorder.

Thus, the determination may comprise: i) conducting or performing a genome or genetic analysis of the individual; and ii) preparing a pharmacogenomic or metabolomic profile and/or identity based on personal and/or clinical information from or about the individual; iii) optionally, performing a test or assay (such as on a biological sample from the individual) that can indicate susceptibility (to age-related macular degeneration).

In (i), this may involve determining an individual's genotype (for the respective loci). It can comprise identifying a haplotype and/or allelic variant (or combination of SNPs), a polymorphism (such as an SNP) or genetic predisposition (to the relevant disorder), as previously described. One may therefore be able to draw up a genetic profile of the individual, preferably relevant to the disorder.

The determination may, alternatively or in addition to (ii), comprise obtaining relevant information from, or about, that individual. That information may be personal and/or clinical information. The information may relate, directly or indirectly, to age- related macular degeneration (or an age-related macular degeneration-related disorder). Such information may comprise information concerning lifestyle, health, nutritional status, diet. Other personal information of relevance can include age, sex, weight and/or ethnic background. Clinical information may comprise current drug and/or vitamin regimes, current or past treatments, familial data, health risks, family background, medical conditions and/or allergies. It may therefore involve obtaining a patient's medical history, or current health status and determining their nutritional profile. The individual may be able to provide this information, for example, by completing a questionnaire.

Determining risk/susceptibility (i) Genetics

In one embodiment of the present invention the identification of the individual risk of a subject of developing AMD, or suffering from AMD (which may have been undetected so far) can be accomplished by genetic (or genome) analysis, more specifically, by determining the presence of gene polymorphism involved in (the development of) or related to AMD, as described earlier.

Such a polymorphism may be present (or near, or associated with) a gene coding for a dehydrogenase, oxgenase, acyltransferase or dismutase, such as a mammalian (e.g. human) dehydrogenase. Preferably it is a retinaldehyde and/or retinal/retinol dehydrogenase. If the enzyme is an oxygenase then it may be a mono or di-oxygenase, such as one associated with carotenoid or macular or retinal pigments, eg. β-carotene.

Preferred acyltransferases are lecithin related eg. lecithin-cholesterol or lecithin-retinol. Of the dismutases, super oxide is preferred.

The applicant has found that these particular SNPs are particularly useful for identifying patients with, or who may develop, AMD. The SNPs therefore represent a selection that allows the identification of patients that are likely to benefit from, and respond to, treatment of AMD. It allows the identification of a subset of patients that may be able to respond to AMD treatment, or least have predisposition to AMD, so that this potential condition can be treated early.

The presence of a polymorphism in a gene, for example encoding a protein which is related to the development of AMD, can be determined by methods known in the art. In general, this involves the extraction of genomic DNA by standard procedures (Sambrook J, Fritsch EF, and Maniatis T.: Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Press; 1989) from blood cells, or buccal mucosa cells, hair cells or any other DNA containing human tissues which are easily and usually non-invasively accessible. Alternatively, commercial kits can be used (i.e. QIA AMP blood kit, Qiagen or any other commercially available DNA extraction kit). The characterization of an individual's genotype, at a given locus, which relates to the determination of the variants or polymorphisms, can be performed according to Standard procedures. These classic technologies (Cotton RGH, Mutation detection, Oxford, Oxford University Press, 1997) that relate to the determination of the polymorphisms include DNA sequencing using 96-channel capillary sequencers, single-strand conformation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis using the partial melting behaviour of double-stranded DNA, heteroduplex analysis involving denaturing high-performance liquid chromatography, chemical or enzymatic cleavage of mismatch pairing method, and mutation detection by coupled transcription-translation (protein truncation test) procedures involving non-sense stop codons. The more recent techniques are real-time PCR methods like TaqMan, mass spectrometry involving single-stranded PCR fragments generated by the dideoxy- nucleotide PCR methodology, DNA MicroArray technology detecting SNPs provided by commercial suppliers (e.g. Affymetrix Inc., Santa Clara, California (CA), Illumina, San Diego, CA and Sequenom, San Diego, CA), and many more.

Table 1 , showing the SNPs used in the present invention, follows.

Table 1

Table 2 below correlates the SEQ ID NOs: in the attached sequence listing with the polymorphisms of Table 1.

Table 2

U) Optically

In a further embodiment of the invention, the determination or identification of the individual risk or susceptibility of a subject of developing AMD or suffering from AMD can, in addition to the genetic (eg. SNP) analysis or technique as described earlier, be accomplished optically, such as by measuring optical density, e.g. of a macular pigment or other suitable optical measurement in or of the eye (e.g the retina). This can comprise measuring the level of a carotenoid, usually a macular carotenoid (such as lutein or zeaxanthin) in the eye of the individual.

This can be accomplished by different methods (Berendschot T and Norren Dv, , Arch Biochem Biophys 2004, 430:149-155, Trieschmann et al, Graefe's Arch. Clin. Exp. Ophthalmol., DOI 10.1007, 2006, Springer- Verlag), for example Heterochromatic Flicker ( (Delori FC, J Opt Soc Am A Opt Image Sci Vis. 2001, 18(6): 1212-30)), Scanning Laser Ophthalmoscopy (SLO), Fundus Reflectometry, Raman Spectroscopy (Ermakov I, J Biomed Opt. 2004 Jan-Feb;9(l): 139-48) or US 5,873,831 (Bernstein/University of Utah).

(a) In a preferred embodiment of the invention, the (profile of) macular pigment optical density (MPOD) can be measured, e.g. by a technique which is based on the method described by Delori et al. (Delori FC et al., J Opt Soc Am A Opt Image Sci Vis. 2001, 18(6): 1212-30). This technique can record spatial profiles of the density of the yellow macular pigment across the retina. On a visual display, subjects view a target that alternates between two spectrally different components. One component, the blue light, is absorbed by the macular pigment, whilst the other component, which appears orange to the eye, is not absorbed by the macular pigment. This differential absorption causes an imbalance between the luminance of these two components and in turn causes the test stimulus to appear to be flickering. Flicker can be eliminated by increasing the luminance of the blue component to compensate for the absorption by the macular pigment, and the lowest luminance just required for this condition is a quantitative measure of MPOD. For the construction of MPOD profiles these measurements are done with the test target presented at different points across the retina. (b) The heterochromatic flicker photometry (HCF) technique uses a visual display and provides a rapid and convenient macular assessment profile (MAP) test. The MAP test is based on the use of an optical notch filter to separate the outputs of the three phosphors of the display into two components, one that is absorbed maximally by the macular pigment (MP) and is derived only from the blue gun (ie., the test beam) and the other that is based on a combination of red and green phosphor luminances and consists largely of long wavelength light that is not absorbed by the MP (i.e., the reference beam). The luminance of the reference beam is 20 cd m-2 and its modulation depth is fixed at 20%. The MAP test makes full use of the advantages of visual displays to produce stimuli of varying size at a number of randomised locations, to generate counter-phased sinusoidal modulation of the two stimulus beams. The frame rate of the display is 140 Hz and the stimulus modulation frequency is 20 Hz. The high temporal modulation frequency employed ensures that at threshold one isolates the activity of luminance flicker detection mechanisms that rely only on the combined L and M cone signals. The stimulus is presented as a short burst of flicker of approximately 0.5s duration and the subject's task was to report the presence or the absence of perceived flicker.

A modified staircase procedure with variable step sizes is then used to measure the mean luminance of the test beam needed to cancel the perception of flicker generated by the reference beam. The MAP test can be used to measure MPOD along any meridian at a number of specified locations from -8° to +8° eccentricity of the visual field. The test stimulus changes from a disc of 0.36 o diameter, when presented at the fovea, to a sector annulus when presented at one of five discrete locations on either side of fixation across the horizontal meridian: ±8°, ±6°, ±4°, ±2.5°, ±1.25°, 0°. The width of the test annulus also increases systematically with eccentricity to facilitate the detection and the nulling of luminance flicker. A central spot and radial guides are used to help the subject maintain steady fixation. Five, randomly interleaved, repeat measurements were taken at each spatial location investigated. The test can be performed at a viewing distance of 0.7m and the stimulus was presented only to the right eye. Similar measurements can be made with the left eye to confirm previous findings to show good correlation in MPOD values between the two eyes.

(c) An optical density of the macular pigment of lower than 0.2 as determined by HCF can be regarded as evidence for an existing risk of developing AMD or the existence of a macular degeneration. This may lead to the administration of a carotenoid, such as lutein, zeaxanthin and/or meso-zeazeanthin, and/or one of the antioxidants or mixture thereof as defined earlier according to step (b) of the method of the present invention (Aleman TS, Duncan JL, Bieber ML, et al. Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome; Invest. Ophthalmol. Vis. Sci. 2001 ;42(8): 1873- 81; Curran-Celentano J, Hammond BR, Ciulla TA, et al. Relation between dietary intake, serum concentrations, and retinal concentrations of lutein and zeaxanthin in adults in a Midwest population, Am J Clin Nutr 2001 ;74(6):796-802; Koh HH, Murray IJ, Nolan D, et al. Plasma and macular responses to lutein supplement in subjects with and without age- related maculopathy: a pilot study. Exp. Eye Res. 2004;79(l):21-27).

(d) In still another embodiment of the present invention the identification of the individual risk of a subject of developing AMD or suffering from known or undetected AMD is accomplished by determining the xanthophyll and/or carotenoid level in a body fluid, such as blood or plasma, and/or skin. The xanthophyll and carotenoid level in plasma and/or skin can be determined by methods known in the art. For example, blood (approx.10 to 15 ml) is collected into pre-cooled Monovettes containing EDTA, and plasma prepared by centrifugation. The preparation of plasma has to be done under appropriate shielding from light. After collection, plasma samples can be stored at -35 ⁰C in the dark. Analyses of xanthophylls and carotenoids is performed by high pressure liquid chromatography according to published protocols (Hartmann D, et al., Am J Clin Nutr 2004;79:410 -7, Aebischer CP, et al., Methods Enzymol 1999;299:348-62.)

Xanthophyll and carotenoid plasma levels below 0.25 μmol/L, measured as described above, may be regarded as indicative for an existing risk of developing AMD or of the existence of a macular degeneration, which may require the (or benefit from) administration of lutein or zeaxanthin to the individual.

(e) A further method of determining risk or susceptibility to AMD involves assessing visual (which includes contrast) performance (such as acuity). The method may comprise detecting the response or reaction of the individual, or the individual's eye, to a visual stimulus, for example in a certain or specific colour, normally in the visible spectrum, for example blue or green light. The light or visual stimulus may be provided at two or more intensities or contrast levels. The (visible) light may be green or blue and/or may have a wavelength within 495 - 535 nm or within 460 - 495 nm. The method may involve assessing the level of the pigment, which may be a macular pigment, preferably one that absorbs the visible light. It may comprise detecting a reaction or response of the individual to a visual display or a visual stimulus, for example where two different intensities or contrast levels of the light provided, and the reaction or response to two or more (or each) intensity or contrast level can be assessed. In a preferred embodiment the individual is shown a display with one or more rings, such as Landolt rings, which may be blue in color and/or have a wavelength of from 470 - 800 nm. This method is described in more detail in European Patent Application Number 06255800.2, filed on 13 November 2006 in the name of DSM IP Assets B. V., the contents of which are herein incorporated by reference.

Sampling

The determination may comprise taking (a biological) sample from the individual, such as a body fluid (such as urine, saliva or blood) that may contain cells. Preferably, a sample may comprise buccal and/or skin cells, for example taken from the mouth using for example a swab.

The genetic analysis may be performed using a microarray (SNPs or one or more genes on a chip) or a multiwell plate, for example in a laboratory. It may thus involve the use of a gene/DNA chip, or a strip, test tube or other solid surface comprising one or more

SNPs or genetic polymorphisms that may or may not cover one or more genes, but are found in a genetic locus of interest.

Communication of substance The nature or identity of the substance can be communicated either to the individual, or their doctor, optician, physician, guardian, dietician or (genetic) counselor. The communication may be electronically, for example via a computer (a personal computer or a laptop), portable computer or mobile phone or using the internet (e.g. via email, webpage and/or blog). Alternatively, it may be communicated on paper, for example in a booklet or information pack.

The communication of the nature or identity of the substance may be provided through a handheld and/or portable device such as a bracelet, necklace or watch-type device, personal computer, cell or mobile phone, personal digital assistant (PDA), a device which may be attached to or integral with a shopping cart or trolley, a terminal (eg. to an on-line service, which may be in an outlet or retail store, such as a super-or hypermarket, pharmacy or health store), for example through the internet, a telephone with voice communication, kiosk or centralised computer system. Genetic determination of susceptibility

The presence of a polymorphism in genes can be determined by methods known in the art, and in general, this will usually involve the extraction of genomic DNA by standard procedures, for example from blood cells, or buccal mucosa cells, hair cells or any other DNA containing tissue, which is suitably easily, and usually non-invasively (or minimally non-invasively) , accessible. Alternatively, commercially available DNA extraction kits can be employed.

The characterisation of an individual's genotype, for a given locus, which may relate to the determination of variance of polymorphisms, can be performed according to standard procedures. This technology can involve the use of DNA sequencing apparatus, for example using a 96-channel capillary sequencer, a single strand conformation analysis using non-denaturing gel electrophoresis, denaturing gradient gel electrophoresis (using the partial melting behaviour of double stranded DNA), heteroduplex analysis involving denaturing HPLC, chemical or enzymatic cleavage of mismatch pairing method and/or mutation detection by coupled transcription-translation (protein truncation test) procedures, for example involving non-sense mutations and/or variations. The most recent techniques are real-time PCR methods like TaqMan mass spectrometry involving a single-stranded PCR fragments, for example generated by the dideoxy-nucleotide PCR methodology, DNA microarray technology for detecting SNPs, as provided by commercial suppliers (such as Affymetrix Inc., Santa Clara, California (CA), Illumina, San Diego, CA and Sequenom, San Diego, CA) and many more.

Substances and compositions to be provided to the individual

The substance or composition may comprise a compound, such as an active ingredient, a drug, pharmaceutical or nutraceutical. The substance may be edible and/or comprise a food, foodstuff or feed, for example a (dietary) supplement, or pharmaceutical composition.

The substance (or composition) may be in any form, for example suitable for oral administration, such as in solid form such as tablets, including effervescent tablets, soft or hard-shell capsules, or in liquid form such as solutions or suspensions, such as an oily suspension. Besides any active ingredient, the preparation may contain one or more conventional (eg pharmaceutical) carrier materials, additives and adjuvants, for example, including one or more of gelatine, vegetable gum, sugar, vegetable oil, polyalkylene glycol, flavouring agent, preservative, stabilizer, an emulsifying agent and/or a buffer. The substance, if medicament, can be a controlled (or delayed) release formulation.

The (therapeutic) substance may be administered in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, and subcutaneously. The pharmaceutical compositions that contain the therapeutic agent will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solids, for example tablets or capsules, or liquid solutions or suspensions. In a preferred embodiment, the therapeutic agent is administered to the individual in their diet, for example in a drink or food. The present invention may thus provide an optimisation of diet and or nutritional supplementation and or pharmaceutical administration, based on the determination of susceptibility to the relevant disorder. The optimisation, for example of nutrition or nutritional supplementation, may be for a group of individuals, usually related ones, such as a family. If the substance is a nutritional supplement, this may include foods, capsules, pills, powders, gums and liquids or other oral dosage forms. Also encompassed are nutritional supplements that can be delivered for example to the digestive system, or intravenously, as well as supplements that can be administered through other routes, such as mucous membranes. The individual supplements may comprise excipients, impurities or other components other than the substance of interest.

Once the individual's susceptibility has been determined, one can optimise the nutritional intake, in particular of the substance or composition, hi this sense the identity of the substance itself, the amount, dosage and the form in which it is ingested or administered can be tailored to that individual, so that the substance is personalised for that particular individual. The result of the examination may include a proposal to reduce intake of supplement, macronutrient or foodstuff, as well as increasing or adding a substance or other nutritional substances. Once the individual has been identified as having (a risk of developing) AMD, or is suffering from known or undetected AMD, an effective amount of a (preferably macular) carotenoid, such as lutein and/or zeaxanthin and/or the AREDS cocktail (a component thereof) can be suggested or administered. The substance can be a xanthophyll (for example, a carotenoid possessing one or more oxygen atoms, such as an - OH or hydroxy group).

An effective amount of the carotenoid can be used. Preferably this is lutein, meso- zeaxanthin, and/or zeaxanthin and/or "the AREDS cocktail" (vitamin C, vitamin E, beta carotene, zinc and/or copper, AREDS Report No. 8, Arch. Ophthalmol. 2001;l 19:1417- 1436, referred to as "AREDS Cocktail", also at HKJ Ophthalmol. Vol. 4, Nr. 1 , (2000), p. 31-42) and/or one of the components of the AREDS cocktail. For the purposes of the present invention this can be e.g., within the range of from 0.001 mg per kg body weight to about 20 mg per kg body weight. More preferred is a daily dosage of about 0.01 to about 10 mg per kg body weight, and especially preferred is about 0.1 to 1.0 mg per kg body weight per day, especially for the carotenoid, e.g. lutein and/or zeaxanthin. Preferably the carotenoid, e.g. lutein and/or (optionally meso-)zeaxanthin are administered at a dosage of from 1 or 5 to 15, 30 or 50 mg/day, such as from 8 or 10 to 12, 15 or 20mg/day and may be present in compositions at that (daily) dosage. Preferred compositions can contain from 8 to 12mg lutein or zeaxanthin (and preferably both within this range). Especially preferred for vitamin C is 1 to about 10 mg per kg body weight, for beta- carotene 0.1 to about 0.3 mg per kg body weight, for vitamin E 1IU to about 10 IU per kg body weight, for zinc 0.1 mg per kg body weight to about 1.5 mg kg body weight, and for copper 0.01 mg per kg body weight to about 0.05 mg per kg body weight. Zinc is preferably used as zinc oxide and copper as cupric oxide. Preferable daily dosages and/or amount in an oral (e.g. daily) formulation, such as a tablet, are as follows. The formulation may comprise an antioxidant. This may be vitamin C (such as at from 200 to 800mg, 400 to 600mg, such as 450 to 550mg). There may be 1, 2 or 3 antioxidants present. In addition or alternatively, another antioxidant is vitamin E. This may be present at a dosage of from 100 to 700 IU, such as from 200 to 600 IU, preferably from 300 to 500 IU. Another preferred antioxidant, instead of or in addition to vitamins C and E, is beta-carotene. Beta-carotene may be present at from 5 to 40mg, such as from 10 or 20 to 30 or 40mg, preferably from 13 to 18 mg. The zinc may be present as zinc oxide, and can be an amount of from 20 to 140mg, such as from 60 to lOOmg, preferably from 70 to 90mg. The copper may be present at from 1 to 2mg.

The duration of the treatment can be suitably life-long, and no shorter than the above- mentioned markers would indicate or suggest that the subject involved is no longer at risk for developing AMD or no longer suffers from AMD. Suitably, treatment is started with an initial dosage of 0.5 - l.Omg of carotenoid (eg. xanthophyll), such as lutein and/or zeaxanthin, per kg body weight per day for 1-2 months whereupon the dosage may be . lower to secure a macular pigment optical density of three times the threshold value, i.e. 0.6. Preferably, two or more xanthophylls are present, such as a combination of lutein and zeaxanthin. In such combination these compounds are preferably used in a ratio of 0.1-1.0 : 1.0- 0.1 parts (by weight), such as from 0.5-1.0:1.0-0.5, especially 0.9-1.1 :0.9-1.1, to each other.

In accordance with the invention, the substance, such as lutein and/or zeaxanthin and/or the "AREDS Cocktail" or its individual components can be provided in any appropriate form, suitably for oral administration, e.g. as a pharmaceutical composition, or in food or beverage. The term "providing" as used herein is to be understood as denoting the act of collecting the desired active ingredients and processing them into a suitable administration form, as well as the direction for use and/or administration to the subject involved. Higher dosages and amounts can be provided to individuals who appear to be at greater risk, for example one or more polymorphisms associated or related to AMD, and so one can correlate higher dosages with greater risk (or more polymorphisms).

In still another aspect, the invention relates to the use of a (preferably macular) carotenoid, e.g. xanthophyll, such as lutein, meso-zeaxanthin: and/or zeaxanthin and/or a vitamin C, beta-carotene, vitamin E, zinc and copper or a mixture thereof in the manufacture of a medicament for the treatment and/or prevention of age-related macular degeneration (AMD) in a subject which has been identified as being at risk of developing AMD, or as suffering from AMD, especially by one of the methods (of the invention) identified above. The nature of the substance (or its dosage for example), may be modified over time.

The substance and/or dosage may therefore change, depending on the individual's response to the treatment, to the subtance, or the nature or condition of AMD. The individual may be able to measure his/her response or reaction to the substance, for example the benefit of the substance administered, or of on going supplements. The individual, patient, physician, optician or other person may therefore change the substance, medicament or other composition, in view of variables such as increasing age, susceptibility to AMD and the progression (or otherwise) of AMD. The individual may therefore be able to self- monitor his/her response to the prevention or treatment that is ocurring.

Databases and foods/compositions

In the determining the susceptibility of an individual, one can obtain personal data, which may be obtained through automated data analysis, interview survey subjective analysis and/or laboratory testing. A database can be provided with information concerning available nutritional supplements, including contents, price and dosage form. A further database may include information, including risks and benefits, about constituency of nutritional supplements, for example information concerning the substance.

The invention can further include apparatus for formulating the substance, for example in a food or in a nutritional supplement, usually based on the determination of susceptibility. A specific formulation may then be provided or communicated to the individual, which may or may not be standard dosage form. The invention thus additionally contemplates a vending machine or point of sale dispensing machine which can formulate, or combine, pre-prepared dosage forms of nutritional supplements, based on the opposed nutritional supplementation or the substance to be taken by the individual. Where the point of sale dispensing machine is in a public location, an interface may be provided, for example a touch screen. Optionally, an individual may be interviewed, optionally in the presence of a trained professional, with the data inputted or accepted in an appropriate format. Thus, a trained professional, such as doctor, nurse, chiropractor, social worker or nutritionist may assist in the input in medical information, etc.

Screening for (therapeutic) substances In one embodiment the invention provides a method for identifying a substance useful for the treatment of age-related macular degeneration, which method comprises contacting a variant age-related macular degeneration polypeptide or a polynucleotide with a test agent and determining whether the agent is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide. Any suitable binding assay format can be used to determine whether the age-related macular degeneration variant binds the test agent, such as the formats discussed below. The method may be carried out in vitro, either inside or outside a cell, or in vivo. In one embodiment the method is carried out on a cell, cell culture or cell extract that comprises a variant age-related macular degeneration protein or polynucleotide. The cell may be any suitable cell, and is typically a cell in which the product is naturally expressed. The term "modulate" includes any of the ways mentioned herein in which the agent is able to modulate activity of an age-related macular degeneration variant polypeptide or polynucleotide. This may be determined by contacting the polypeptide or polynucleotide with the test agent under conditions that permit activity of the polypeptide or polynucleotide, and determining whether the test agent is able to modulate the activity of the polypeptide or polynucleotide. In one aspect of the invention, the test agent is a food ingredient. Hence, the invention relates to a method of screening food ingredients to determine whether they contribute to or aggravate age-related macular degeneration in susceptible individuals, or if they prevent or alleviate age-related macular degeneration.

The present invention also provides an agent identified by a screening method of the invention. An agent identified in the screening method of the invention may be used in the therapeutic treatment of a age-related macular degeneration. Such an agent may be formulated and administered in any means or amounts as discussed below.

Customised composition (eg. food) In one aspect, the invention relates to a customised diet for an individual that is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder).

Accordingly, the present invention enables the preparation of a customised composition (or diet) suitable for an individual who is susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder), wherein the customised composition or diet comprises one or more ingredient(s) that can prevent or alleviate age-related macular degeneration (or age-related macular degeneration-related disorders) and/or does not comprise components that contribute to or aggravate age-related macular degeneration (or age-related macular degeneration-related disorders). Such ingredients may be any of those known in the art to prevent or alleviate age-related macular degeneration. Alternatively, screening methods as discussed herein may identify such ingredients. The preparation of customised food may be carried out using electronic means, for example by using a computer system.

In one embodiment, the composition may be formulated to alter the profile of food proteins in order to minimise the potential for secondary dietary sensitivity. The customised food may be hypoallergenic and/or may exclude ingredients that are poorly tolerated or cause allergies, for example gluten-containing grains such as wheat, particular protein sources such as animal proteins, milk (lactose), eggs, soy, peanuts, shellfish, fruits or tree nuts.

In another embodiment, the (customised) composition may be formulated to include functional or active ingredients that help prevent or alleviate age-related macular degeneration (or an age-related macular degeneration-related disorder).

The present invention also relates to a method of providing a composition (eg. food) suitable for an individual who has been determined to be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder) such as by a method of the invention. The customised composition can be made to an inventory and supplied from inventory, i.e. is pre-manufactured rather than being made to order. Therefore the composition may not be specifically designed for one particular individual but may be suitable for a relative of the individual that may also be susceptible to age-related macular degeneration (or an age-related macular degeneration-related disorder). Alternatively, the composition may be suitable for a group of individuals that are susceptible to an age- related macular degeneration-related disorder, such as members of a family, hi preferred embodiment, the composition is personalised or customised to meet the nutritional requirements of a specific individual. Bioinformatics

The sequences of the age-related macular degeneration variants or SNPs may be stored in an electronic format, for example in a computer database. Accordingly, the invention provides a database comprising information relating to age-related macular degeneration allelic variant sequences, which may include further information about the allelic variant, for example the level of association of the allelic variant with an age-related macular degeneration-related disorder or the frequency of the allelic variant in the population. The database can comprise information regarding the substance(s), which are suitable and/or not suitable for individuals (e.g. who may possess a particular allelic variant of age-related macular degeneration).

A database may be used to determine the susceptibility of an individual to age-related macular degeneration (or an age-related macular degeneration-related disorder). Such a determination may be carried out by electronic means, for example by using a computer system (such as a PC). Typically, the determination will be carried out by inputting genetic data from the individual to a computer system; comparing the genetic data to a database comprising information relating to age-related macular degeneration allelic variants; and on the basis of this comparison, determining the susceptibility of the individual to AMD or an age-related macular degeneration-related disorder.

The invention also provides a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer. Also provided is a computer program product comprising program code means stored on a computer readable medium for performing a method of the invention when said program is run on a computer. A computer program product comprising program code means on a carrier wave that, when executed on a computer system, instruct the computer system to perform a method of the invention is additionally provided.

The invention also provides an apparatus arranged to perform a method according to the invention. The apparatus typically comprises a computer system, such as a PC. In one embodiment, the computer system comprises: means for receiving genetic data from the individual; a module for comparing the data with a database comprising information relating to age-related macular degeneration allelic variants; and means for determining on the basis of said comparison the susceptibility of the individual to an age-related macular degeneration-related disorder. The invention may also use an advice decision tree wherein the results of the genetic polymorphism analysis may be used to correlate the genetic profile of susceptible individual together with the identified risk factors based on nutrition and life-style questionnaires. Both information sources may be used to generate advice for treatment or risk reduction/prevention of age-related macula degeneration or age-related macular degeneration-related diseases. In generating the advice, other factors concerning the sex and health of the individual and or of the individual's family, age, alcohol consumption and existing diet may be used as well in the determination of appropriate lifestyle and nutritional recommendations.

Composition/food manufacturing

In one embodiment of the invention, the manufacture of a customised composition may be controlled electronically. Typically, information relating to the age-related macular degeneration allelic variant(s) present in an individual may be processed electronically to generate a customised composition. The customised composition may then be used to generate electronic manufacturing instructions to control the operation of composition manufacturing apparatus.

The apparatus used to carry out these steps will typically comprise a computer system, such as a PC, which comprises means for processing the nutritional information to generate a customised composition; means for generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus; and a composition manufacturing apparatus.

The composition manufacturing apparatus may comprise a packaging apparatus. The packaging apparatus typically packages the composition into a container (such as a plastic or paper bag or box). The apparatus may also comprise means for labelling the composition, typically after packaging. The label may provide information such as: ingredient list; nutritional information; date of manufacture; best before date; weight; and types of individual(s) for which the composition is suitable.

Preferred features and/or characteristics of one aspect of the invention are applicable to another aspect mutatis mutandis. The invention is illustrated further by the Examples given below: Example 1

From an adult individual a buccal (cheek) swab using a fibre brush or a Q-tip is taken. The buccal (cheek) swab is stored at 4⁰C until analysis. The buccal mucosa cells derived from this swab are used for DNA analysis and determination of the genotype. DNA extraction is performed according to commercial suppliers (e.g. Qiagen Ltd, 8634 Hombrechtikon, Switzerland) using standardized protocols e.g. "Isolation of DNA from buccal cells using the EZl DNA Tissue Kit (Qiagen Ltd, 8634 Hombrechtikon, Switzerland)". This protocol is designed for the isolation of total genomic and mitochondrial DNA from buccal cells. The genotype analysis can be performed involving diverse technologies which are known to a skilled person and which are available through commercial services.

The following genotypes as a risk factor is included in the analysis: a) the SNP rs6657239; and b) the SNP rs 1800549.

The identification of the haplotypes for the specific genes listed follows the procedures in the respective references cited for each gene. The risk evaluation according to step (a) of the claimed method may involve one or more of the individual methods discussed above, i.e., by genome or genetic analysis and/or determining the macular pigment optical density and/or xanthophyll and carotenoid plasma level.

Example 2

A buccal (cheek) swab was taken from an individual (adult man, aged 60, different from the individual in Example 1) and DNA extracted as described in Example 1. The DNA is analysed for the SNPs rs 7123686 and rs 2676377. The adult was found to have both of these polymorphisms, and was recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.

Example 3 A buccal (cheek) swab was taken from an individual (adult man, aged 56) and DNA extracted as described in Example 1. The DNA is analysed for the four SNPs rs 3118416, rs 2182315, rs 3118418 and rs 12145904. The adult was found to have two of these polymorphisms, and was recommended a course of zeaxanthin at a dosage of 12mg/day, reducing to 6mg/day after one month.

Examples 4 and 5 Soft gelatin capsules to be administered to an individual were prepared comprising the following ingredients:

Ingredient Amount per Capsule

Lutein 8 and 10 mg

Lecithin 50 mg Soy bean oil 200 mg

One or more capsules may be taken, suitably with breakfast.

Examples 6 and 7

Soft gelatin capsules were prepared comprisi

Ingredient Amount per Capsule

Lutein 8 and 10 mg

Zeaxanthin 8 and 10 mg

Lecithin 50 mg

Soy bean oil 200 mg

Examples 8 and 9

Soft gelatin capsules were prepared comprising the following ingredients: Ingredient Amount per Capsule

Lutein 6 mg Zeaxanthin (or meso-zeaxanthin) 6 mg

Lecithin 50 mg

Soy bean oil 200 mg Example 10

Lutein 12 mg Lecithin 50 mg

Soy bean oil 200 mg

Example 11

Soft gelatin capsules was prepared comprising the following ingredients: Ingredient Amount per Capsule

Zeaxanthin 12 mg

Lecithin 50 mg

Soy bean oil 200 mg

Example 12

Soft gelatin capsules were prepared comprising the following ingredients:

Ingredient Amount per Capsule

.Lutein 12 mg

Zeaxanthin 12 mg Lecithin 50 mg

Soy bean oil 200 mg

Examples 13 to 19

Eight soft gelatin capsules were prepared comprising the following ingredients:

Claims

1. A method of determining whether (or not) an individual is susceptible to or has acute macular degeneration (AMD), or an AMD - related disorder, the method comprising typing the nucleotide present in the genome of the individual for the presence of one or more polymorphism(s), or detecting the presence of one or more polymorphism(s) in sample (comprising DNA) from the individual, wherein the polymorphism is as identified in Table 1 , or is a polymorphism associated with a dehydrogenase, oxygenase, acyltransferase or dismutase or a macular or retinal pigment, and/or at one or more positions which are in linkage disequilibrium with any one of these positions.

2. The method according to claim 1, wherein the polymorphisms from Table 1 are one or more of the 7 polymorphisms related to: a) ABCA4 of Table 1 , eg. rs6657239 or rsl 800549; b) BCDO2 of Table 1, eg. rs7123686 or rs2676377; c) CST3 of Table 1, eg. rsl 1504 or rsl 135147; d) FBLN5 of Table 1, eg. rs929608, rs2430347, rs 2474028 or rsl7127768; e) ILIA of Table 1, eg. rsl304037, rsl7561, rs2856841 or rs2856837; f) NFE2L2 of Table 1, eg. rs2364731, rs7568033, rs6433659, or rs3813259; g) RPE65 of Table 1, eg. rs3118416, rs2182315, rs3118418 or rsl2145904; h) SODl of Table 1, eg. rs3216079; i) TF of Table 2, eg. rsl 130459, rsl 2769, rsl 800669 or rs8649.

3. The method according to any one of the preceding claims, wherein the polymorphisms are: a) ABCA4 of Table 1, eg. rs6657239 or rsl800549; b) BCDO2 of Table 1, eg. rs7123686 orrs2676377; c) RPE65 of Table 1, eg. rs3118416, rs2182315, rs3118418 or rsl2145904; d) SODl of Table 1, eg. rs3216079.

4. The method according to any preceding claims, wherein the polymorphisms are: a) rs6657239 or rsl 800549.

5. A method according to any one of the preceding claims wherein the polymorphism is associated with a gene encoding a dehydrogenase, oxygenase or acytransferase.

6. The method according to any preceding claim wherein the polymorphism is associated with a gene encoding a retinaldehyde and/or retinal/retinol dehydrogenase, a mono-or di-oxygenase or a gene related to or associated with a macular or retinal pigment or a carotenoid.

7. A method for treatment and/or prevention of age-related macular degeneration

(AMD), which method comprises: a) conducting a method according to any preceding claim; and b) providing (an effective amount of) a substance to the individual, wherein the substance is able to prevent or treat age-related macular degeneration, or mitigate or alleviate symptoms of age-related macular degeneration (AMD).

8. A method according to claim 7 wherein the substance comprises (an effective amount of) a carotenoid and/or vitamin C, vitamin E; beta carotene, zinc and/or copper, and/or or a mixture thereof (the AREDS Cocktail) to said subject.

9. A method of claim 7 or 8 wherein the carotenoid is lutein, zeaxanthin and/or meso-zeaxanthin.

10. A method according to any of claims 7 to 9 wherein the substance is a carotenoid, such as a xanthophyll.

11. A method according to any of claims 7 to 10 wherein, in (b) the substance is provided at from 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg of carotenoid (lutein and/or zeaxanthin) per kg body weight per day.

12. A method according to any of claims 7 to 11 wherein, in (b) the substance is provided at from 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg per kg body weight of lutein and/or zeaxanthin plus vitamin C (1 to about 10 mg per kg body weight), beta- carotene (0.1 to about 0.3 mg per kg body weight), vitamin E (1IU to about 10 IU per kg body weight), zinc (0.1 mg per kg body weight to about 1.5 mg kg body weight), copper (0.01 mg per kg body weight to about 0.05 mg per kg body weight) per day.

13. (The use of) a substance that is able to prevent or treat AMD or mitigate or alleviate symptoms of AMD, optionally in the manufacture of a medicament, for the treatment and/or prevention of age-related macular degeneration (AMD) in a subject who, usually on the basis of a sample taken therefrom, has been subjected to the determination method of any of claims 1 to 6.

14. The use according to claim 13 wherein the polymorphism is as defined in any one of claims 1 to 6 and/or the substance comprises a carotenoid, such as lutein, meso- zeaxanthin and/or zeaxanthin, and/or a mixture of vitamin C, beta-carotene, vitamin E, zinc and copper.

15. The use according to claim 13 or 14 wherein the medicament contains an amount of carotenoid, such as lutein/and or zeaxanthin, which is sufficient to administer 0.001 mg to 20 mg, preferably 0.1 mg to 1.0 mg of lutein and/or zeaxanthin per kg body weight per day.

16. A method of determining a substance to be administered to an individual, the method comprising: a) conducting a method of any of claims 1 to 6; and b) on the basis of (a), identifying a substance capable of treating and/or preventing age-related macular degeneration in that individual.

17. A method according to claim 16 additionally comprising: c) providing (such as administering or communicating) the substance (or its identity) to the individual.

18. A method according to claim 16 or 17 when the determination comprises additionally obtaining relevant information from the individual, such as personal and/or clinical information.

19. A method according to claim 18 when the information comprises personal information on the lifestyle, health, nutritional status, ethnic background, diet, age, sex and/or weight of the individual.

20. A method according to claim 18 when the information comprises clinical or medical information such as current or past vitamin and/or drug regime or treatments, medical conditions and/or any allergies.

21. A method according to any of claims 16 to 20 when the determination comprises taking a biological sample of the individual, such as a body fluid, and/or a sample comprising cells and optionally, additionally analysing a sample for a biomarker as an indicator of age-related macular degeneration.

22. A method according to claim 21 wherein the body fluid is urine, saliva and/or blood and/or the biological sample comprises buccal cells.

23. A method according to any of claims 16 to 22 wherein in (b), a proposal, suggestion or recommendation is made to the individual concerning the substance to be administered.

24. A method according to any of claims 16 to 23 wherein the substance is a compound (such as drug, pharmaceutical, or nutraceutical), a foodstuff, feed or dietary supplement.

25. A method of preparing a customised composition for an individual, the method comprising:

(a) conducting a determination method according to any of claims 1 to 6; and (b) preparing a composition suitable for, or tailored to, the individual.

26. A method according to claim 25, wherein the customised composition comprises ingredients which prevent or alleviate age-related macular degeneration (or an age-related macular degeneration -related disorder) and/or does not comprise ingredients which contribute to or aggravate age-related macular degeneration (or an age-related macular degeneration-related disorder).

27. A method according to claim 25 wherein the customised composition comprises a therapeutic substance.

28. A method of providing a customised composition, the method comprising providing a composition suitable for an individual who has been tested for the presence of a polymorphism by a method according to any of claims 1 to 6.

29. A method for identifying a substance for the treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising:

(a) contacting an age-related macular degeneration allelic variant polypeptide or a polynucleotide, which encodes or is related to an age-related macular degeneration allelic variant or polymorphism, wherein the polymorphism is associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease, with a test agent; and (b) determining whether the agent is capable of binding to the polypeptide or modulating the activity or expression of the polypeptide or polynucleotide; and (c) optionally, providing (such as administering or communicating) the substance (or its identity) to an individual.

30. Use of a compound which is therapeutic for age-related macular degeneration, or an AMD-related disorder (optionally, in the manufacture of a medicament) for the prevention or treatment of age-related macular degeneration (or an age-related macular degeneration-related disorder) in an individual that has been tested for the presence of a polymorphism, wherein the polymorphism associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1 or is related to or associated with a (e.g. high temperature requirement) serine protease.

31. A method of treating an individual for age-related macular degeneration (or an age-related macular degeneration-related disorder), the method comprising administering to the individual (an effective amount of) a therapeutic compound which prevents or treats AMD or the related disorder, wherein the individual has been tested for the presence of a polymorphism, wherein the polymorphism is associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1.

32. A database comprising information relating to age-related macular degeneration polymorphisms, wherein the polymorphism associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1 , and optionally their association with age-related macular degeneration, or age-related macular degeneration-related disorder(s) and/or substances capable of preventing or treating age- related macular degeneration.

33. A method for determining whether an individual is susceptible to age-related macular degeneration (AMD), or an age-related macular degeneration-related disorder, the method comprising: (a) inputting data of one or more age-related macular degeneration allelic polymorphism(s), wherein the polymorphism associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1 ; (b) comparing the data to a computer database, which database comprises information relating to age-related macular degeneration allelic variants and the age-related macular degeneration susceptibility associated with the polymorphism(s); and (c) determining on the basis of the comparison whether the individual is susceptible to age-related macular degeneration (or an age-related macular degeneration- related disorder).

34. A computer program comprising program code means for performing all the steps of claim 33 when said program is run on a computer.

35. A computer program product comprising program code means stored on a computer readable medium for performing the method of claim 16 or 33 when said program product is run on a computer.

36. A computer program product comprising program code means on a carrier wave, which program code means, when executed on a computer system, instruct the computer system to perform a method according to claim 35.

37. A computer system arranged to perform a method according to claim 33 comprising:

(a) means for receiving data of the one or more age-related macular degeneration allelic variant(s) present in the individual;

(b) a module for comparing the data with a database comprising information relating to age-related macular degeneration allelic variants and the age-related macular degeneration susceptibility associated with the variants; and

(c) means for determining on the basis of said comparison whether the individual is susceptible to age-related macular degeneration, or an age-related macular degeneration- related disorder.

38. A method of preparing a customised composition for an individual, the method comprising:

(a) determining whether the individual possesses a polymorphism, wherein the polymorphism is associated with a dehydrogenase, oxygenase, acyltransferase, dismutase or a macular or retinal pigment, or is shown in Table 1 ;

(b) (e.g. electronically) generating a customised composition suitable for the individual;

(c) optionally, generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition; and (d) manufacturing the customised composition (according to the electronic manufacturing instructions).

39. A computer system according to claim 38, further comprising:

(e) means for electronically generating a customised composition formulation suitable for the individual;

(f) means for generating electronic manufacturing instructions to control the operation of composition manufacturing apparatus in accordance with the customised composition; and

(g) a composition product manufacturing apparatus.

40. Use of a computer system as defined in claim 39 to make a customised composition.