JP5290772B2 - Mutants in complement regulatory genes predict age-related macular degeneration - Google Patents

Abstract

Methods for identifying a subject at risk for developing AMD are disclosed, as are kits which can be used to practice the methods. The methods include identifying specific protective or risk polymorphisms or genotypes from the subject's genetic material, including polymorphisms in the BF, C2 and/or CFH genes. Microarrays and kits for use in these methods are also provided.

Description

This application claims the benefit of US Provisional Patent Application No. 60 / 772,989, filed Feb. 13, 2006. US Provisional Patent Application No. 60 / 772,989 is hereby incorporated by reference in its entirety.

FIELD This application relates to a method for predicting a person's genetic susceptibility to age-related macular degeneration.

Acknowledgments for Government Support The present invention is supported by the Government of the United States with grant numbers EY13435 (RA) and EY11515 (GSH) from the National Institutes of Health, and is contract number NO1-CO of the National Institutes of Health National Cancer Institute. -Partly by a US government agency with 124,000 federal grant assistance. The US federal government has certain rights in this invention.

BACKGROUND OF THE INVENTION Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a photoreceptor-rich region in the center of the retina that provides a detailed visual image. AMD usually suddenly worsens central vision without compromising peripheral vision. AMD is the most common form of unrecoverable blindness in developed countries. This disease generally shows a decrease in central vision in one eye, and within a few months to years, the other eye likewise loses central vision. Clinical signs of the disease include the appearance of deposits (drusen) in the macula.

Despite the major health insurance burden, the etiology and pathogenesis of AMD is still poorly understood. Numerous studies have implicated inflammation in the pathobiology of AMD (Anderson et al. (2002) Am. J. Ophthalmol. 134: 411-31; Hageman et al. (2001) Prog. Retin. Eye Res. 20: 705-32; Mullins et al. (2000) Faseb J. 14: 835-46; Johnson et al. (2001) Exp. Eye Res. 73: 887-96; Crabb et al. (2002) Proc. Natl. Sci.U.S.A. 99: 14682-7; Bok, D. (2005) Proc.Natl.Acad.Sci.U.S.A. 102: 7053-4). Complement pathway dysfunction causes severe bystander damage to cells in the macula, resulting in atrophy, degeneration, and production of choroidal neovascular membranes similar to those that occur in other complement-mediated disease processes (Hageman et al., (2005) Proc. Natl. Acad. Sci. USA 102: 7227-32; Morgan and Walport (1991) Immunol. Today 12: 301-6; Kinoshita (1991). ) Immunol.Today 12: 291-5; Holers and Thurman (2004) Mol.Immunol.41: 147-52). Inheritance may be strongly involved in this disease. For example, it is said that variants of FBLN6, ABCA4, and APOE genes are related to risk factors. Recently, it was discovered that a variant of the complement factor H gene (CFH) encoding a major inhibitor of the second complement pathway is associated with an increased risk of developing AMD (Non-Patent Literature). 1; Non-patent document 2; Non-patent document 3; Non-patent document 4).
Haines et al. (2005) Science 308: 419-21. Klein et al. (2005) Science 308: 385-9 Edwards et al. (2005) Science 308: 421-4. Hageman et al. (2005) Proc. Natl. Acad. Sci. U. S. A. 102: 7227-32

  Due to the prevalence of this disease and the limited treatment available, there is a need for a method to identify subjects at risk for developing AMD.

Summary Polymorphisms and genotypes that protect against age-related macular degeneration (AMD) have been identified. Methods are provided for identifying subjects who have an increased risk of developing AMD. These methods analyze a subject's factor B (BF) and / or complement component 2 (C2) gene and determine whether the subject has at least one protective polymorphism Including, but not limited to. Examples of such protective polymorphisms include (a) R32Q in BF (rs641153); (b) L9H in BF (rs4151667); (c) IVS10 in C2 (rs547154); and (d) C2 E318D (rs93332739) and the like. Alternatively, the method can be performed by detecting a protein variant in the body of the subject. If the subject does not have at least one protective polymorphism, the subject has an increased risk of developing AMD. In one embodiment of this aspect, further analysis of the subject CFH gene is performed. In some embodiments, a subject's genotype at the CFH locus can be analyzed to determine whether the subject has at least one protective genotype. In one embodiment, the genotype of a subject at either the BF locus or the C2 locus and the CFH locus can be analyzed to determine whether the subject has at least one protective genotype. . As will be described later in this specification, in some cases it may be helpful to know whether the subject is homozygous or heterozygous for its polymorphism.

  Examples of protective genotypes are: (a) heterozygous for the R32Q (rs641153) polymorphism within BF; (b) heterozygous for the L9H (rs4151667) polymorphism within BF; (c) IVS10 (rs547154) polymorphism within C2. (D) heterozygous for the E318D (rs93332739) polymorphism in C2; (e) homozygous for the delTT polymorphism of CFH; (f) homozygous for the R150R (rs1048709) polymorphism in BF; and (G) Homozygous for Y402 in CFH. If the subject does not have at least one protective genotype, the subject has an increased risk of developing AMD.

The present invention provides a method for assessing the risk of developing macular degeneration or other complement-mediated disease in a human subject, or the likelihood that it is progressing. The background of this method is the discovery made by genetic association studies that link certain genetic features to complement-related diseases, in this case the risk phenotype or age-related macular degeneration . The method of the present invention involves obtaining a biological sample from a human subject and analyzing the sample by any effective technique known in the art to determine whether the subject has one or more of the following: Including the steps of:
A or G in rs641153 of the BF gene: this translates to R or Q at position 32 of the human BF protein;
A or T in rs4151667 of the BF gene: this translates to L or H at position 9 of the human BF protein;
G or T in rs547154 of the C2 gene: this is intron 10;
In rs9332 73 9 of C2 gene C or G: This translates to E or D at position 318 of the human C2 protein;
A or G in rs1048709 of the BF gene: this translates to R at position 150;
DelTT in the CFH gene; and C or T in rs1061170 of the CFH gene: it translates to Y or H at position 402 of the human CFH protein.

In certain embodiments, the sample is analyzed to determine whether the subject has one or more of the following:
A or G in rs641153 of the BF gene: this translates to R or Q at position 32 of the human BF protein;
A or T in rs4151667 of the BF gene: this translates to L or H at position 9 of the human BF protein;
In rs547154 of C2 gene G or T: This is the intron 10; and C2 genes rs9332 73 9 in C or G: This translates to E or D at position 318 of the human C2 protein.

  In some embodiments, the sample is an accessible body fluid, such as blood or blood components, or urine. When the evaluation is performed at the DNA or mRNA level, cell material is required to enable detection of the genotype from the target cell.

  In some embodiments, the subject may have been diagnosed with symptoms such as AMD, early AMD, choroidal neovascularization (CNV), or geographic atrophy (GA). In one embodiment, the subject exhibits symptoms of an early macular degeneration, such as a symptom of the disease, eg, the development of drusen. Some subjects may indicate the occurrence of drusen. The subject may be asymptomatic for macular degeneration or other complement-related disease, in which case the analysis may be for the entire population or for any sub-group that is considered at increased risk, e.g. It essentially provides a screening method that can be performed on individuals with a family history of complement-related diseases. Yet another subject may be at high risk of becoming AMD. In one embodiment, the subject has a Y402H SNP.

  Accordingly, in another aspect, the present invention provides a kit for assessing the risk of developing macular degeneration or other complement-mediated disease in a human subject, or the likelihood that it is progressing. The kit includes a group of reagents for detecting one or more, preferably two or more of the polymorphisms or allelic variants in a sample collected from a subject. The kit can also include oligonucleotides, generally labeled oligonucleotides, designed to detect variants using several methods known in the art. The kit provides a basis for obtaining relevant gene / proteomic information from a sample, eg, PCR primers for amplifying the sequence of the target polynucleotide or, for example, a monoclonal antibody if the target is polymorphic. As a specific binding protein that recognizes and specifically binds to an allelic variant of the target protein. In a preferred embodiment, the kit includes an oligonucleotide immobilized on a solid support.

  Depending on the format, a component of the kit for identifying a subject at high risk of developing age-related macular degeneration (AMD) may include one or more protective polymorphisms to detect at least one protective polymorphism in the subject. Contains reagents. Such reagents allow detection of at least one of the following protective polymorphisms: (a) R32Q in BF (rs641153); (b) L9H in BF (rs4151667); (c) in C2 IVS10 (rs547154); and (d) E318D (rs93332739) in C2. The reagent in such a kit can include one or more oligonucleotides that detect the protective polymorphism. Other kit components can also include one or more reagents for amplifying the target sequence if the target sequence includes one or more of the protective polymorphisms. In some versions of the kit, one or more oligonucleotides are immobilized on a solid support.

  In a related aspect, the invention provides a microarray for identifying subjects at high risk for developing AMD. In a further aspect, the present invention provides a microarray comprising oligonucleotide probes that can hybridize under stringent conditions to one or more nucleic acid molecules having a protective polymorphism. Examples of such protective polymorphisms include (a) R32Q in BF (rs641153); (b) L9H in BF (rs4151667); (c) IVS10 in C2 (rs547154); and (d) C2 E318D (rs93332739) and the like. Such microarrays can further include, for example, oligonucleotide probes that can hybridize under stringent conditions to one or more other nucleic acid molecules having the following polymorphisms: (a) DelTT polymorphism in CFH; (b) R150R polymorphism in BF; and (c) Y402H polymorphism in CFH.

  The above features and advantages of the present disclosure, as well as other features and advantages, will become more apparent from the following detailed description of several embodiments.

Sequences Nucleic acid and amino acid sequences listed in the attached sequence listing are standard abbreviations for nucleotide bases and 3 for amino acids as defined in 37 CFR 1.822. It is shown using a character code. Only one strand of each nucleic acid sequence is shown, but by referring to the displayed strand, it is understood that its complementary strand is also included. Since all accession numbers in the sequence database referred to in this specification were available on the specified date, it means that the version of the sequence identified by that accession number Understood.

  SEQ ID NO: 1 is based on the SNP of refSNP ID: rs641153 that was available via NCBI on January 30, 2006 (modified on January 5, 2006). This SNP gives rise to an R32Q mutant (amino acid at position 32 is glutamine rather than arginine) at position 22 of the BF gene at A or G. The sequence that results in R32Q is CCACTCCATGGTCTTTGGCCCRGCCCCAGGGATCCTGCTCTCT, where R = A or G (SEQ ID NO: 1).

  SEQ ID NO: 2 shows the SNP with refSNP ID: rs4151667 that was available via NCBI on January 30, 2006 (modified on January 5, 2006). This SNP gives rise to an L9H variant (amino acid at position 9 is not leucine but histidine) at nucleotide 26 at position 26 of the BF gene. The sequence resulting in rs4151667 is ATGGGGAGCAATCTCAGCCCCAACRCTGCCTGATGCCCTTTATTCTGGGC, where R = A or T (SEQ ID NO: 2).

  SEQ ID NO: 3 is based on the SNP of refSNP ID: rs547154, which was available via NCBI on January 30, 2006 (modified January 5, 2006). In this SNP, the nucleotide at position 23 of intron 10 of the C2 gene is G or T. The sequence leading to rs547154 is GAGGAGCCCCGCAGAGGCCCGTRTTGGAACCTGGACACAGTGCCC, where R is G or T (SEQ ID NO: 3).

  SEQ ID NO: 4 shows the SNP of refSNP ID: rs93332739 that was available via NCBI on January 30, 2006 (modified January 5, 2006). This SNP gives rise to an E318D variant (amino acid at position 318 is aspartic acid instead of glutamic acid) at position 26 of the C2 gene is C or G. The sequence resulting in rs93332739 is ACGACAACTCCCCGGGATATGACTGARGGTGATCAGCAGCCCTGGAAAATGCCA, where R is C or G (SEQ ID NO: 4).

  SEQ ID NO: 5 shows the SNP of refSNP ID: rs1048709 that was available via NCBI on January 30, 2006 (modified January 5, 2006). In this SNP, the nucleotide at position 26 of the BF gene is A or G. This SNP does not cause an amino acid mutation at position 150 (R150R). The sequence resulting in rs1048709 is ATCGCACCTGCCCAAGTGAATGGCCGRTGGAGTGGGCAGACAGCGATCTTGTG, where R is A or G (SEQ ID NO: 5).

  SEQ ID NOs: 6 and 7 show the delTT polymorphic sequence. The delTT polymorphism is a 2 bp insertion / deletion polymorphism. This sequence is as follows. CCTTGCTATTACATACTAATTCATAACTTTTTTTTTCGTTTTAGAAAGCCCTTGTGGACA (SEQ ID NO: 6) and CCTTGCTATTATACACTAATTCATAACTTTTTTTTTTTTCGTTTTAGAAAGGCCCCTTGGACA (SEQ ID NO: 7).

  SEQ ID NO: 8 shows the SNP of refSNP ID: rs1061170 that was available via NCBI on January 30, 2006 (modified on January 5, 2006). This SNP gives rise to a Y402H variant of the CFH gene (amino acid at position 402 is not tyrosine but histidine) at position 1277 of exon 9 at C or T (nucleotide position 26 in the following sequence). The sequence resulting in rs1061170 is TTTGGAAAAATGGATAATACAAAATRATGGAAGAAAGTTTGTACAGGGTAA, where R is C or T (SEQ ID NO: 8).

  SEQ ID NO: 9 shows the entire amino acid sequence of BF, which is 9H and 32R:

配列番号１３は、３２Ｑである、ＢＦの９個のアミノ酸配列を示している：ｗｓｌａｑｐｑｇｓ（配列番号１３）。

SEQ ID NO: 13 shows the 9 amino acid sequence of BF which is 32Q: wslaqpqgs (SEQ ID NO: 13).

  SEQ ID NO: 14 shows the 9 amino acid sequence of BF, which is 9H: lspqhclmp (SEQ ID NO: 14).

  SEQ ID NO: 15 shows the 7 amino acid sequence of C2, which is 318D: dmtdvis (SEQ ID NO: 15).

DETAILED DESCRIPTION OF THE INVENTION Provided herein are sequence polymorphisms that have been found to be protective against age-related macular degeneration (AMD). These polymorphisms include those found within the factor B (BF) gene and the complement component 2 (C2) gene. Protective polymorphisms also include the delTT polymorphism within the CFH gene. Identifying subjects with these polymorphisms together with subjects with a recently discovered risk haplotype (Y402H in the complement factor H (CFH) gene) diagnoses subjects facing genetic risk of AMD Will help to do.

Terms The following terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in practicing the present disclosure. The singular forms “a”, “an”, and “the” mean one or more than one unless the context clearly indicates otherwise. For example, the term “comprising one nucleic acid” includes one or more nucleic acids and is considered to be identical to the phrase “comprising at least one nucleic acid”. The term “or” means a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes”. Thus, “including A or B” means “including A, B, or A and B”, without excluding further elements. For example, the phrase “mutation or polymorphism” or “one or more mutations or polymorphisms” means one mutation, one polymorphism, or a combination thereof, where “one” is one It can also mean the above.

  Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. These materials, methods, and examples are illustrative only and not limiting.

  Unless otherwise noted, technical terms are used according to conventional usage. General terminology in molecular biology is defined by Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. Science Ltd. Issued 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc. Issue, 1995 (ISBN 1-56081-569-8).

  Age-related macular degeneration: A condition in which light-sensitive cells in the macula become dysfunctional and eventually stop functioning. In macular degeneration, the final form or disease results in a loss or loss of vision in the reader in the middle of the field of view. The perimeter outside the field of view is not compromised. AMD is further divided into “dry” or non-wetting types and “wet” or leachable types. Between 85 and 90 percent of cases are classified as “dry” macular degeneration, in which adipose tissue known as drusen accumulates slowly behind the retina. The classic lesion of dry macular degeneration is map-like atrophy. Ten to fifteen percent of cases are associated with abnormal vascular growth under the retina. These cases are called “wet” macular degeneration because blood and other fluids leak from the back of the retina into the eyeball. Wet macular degeneration usually begins as a dry form. If left untreated, it usually completely destroys the structure and function of the macula. Choroidal neovascularization is a growth of abnormal blood vessels directly under the retinal pigment epithelium (RPF) layer of the retina.

  For wet macular degeneration, drug therapy, photodynamic therapy, laser photocoagulation therapy, and laser therapy can be used. Risk factors for AMD include aging, smoking, family history, exposure to sunlight, especially blue light, cardiovascular risk factors such as high blood pressure, high cholesterol and obesity, high fat intake, oxidative stress, and race. is there.

  AMD is an example of a disease characterized by dysregulation of the second complement cascade, including membranous proliferative glomerulonephritis (MPGN), and a predisposition to develop an aortic aneurysm. The methods described herein for detecting an increased risk of developing AMD are used to detect an increased risk for other diseases characterized by dysregulation of the second complement cascade (eg, MPGN). You can also.

  Allele: Any one of several feasible DNA codes for the same gene occupying a given locus (location) on a chromosome (sometimes this term may mean a non-gene sequence). The individual's genotype for that gene should be the set of alleles it has by chance. In an organism having two copies of each chromosome (diploid organism), the two alleles constitute the individual's genotype. In a diploid organism, if two copies of a gene are identical, that is, if they have the same allele, they are said to be homozygous for that gene. A diploid organism with two different alleles at that gene is said to be heterozygous.

  As used herein, the process of “allelic detection” can be expressed as “genotyping, determining, or identifying an allele or polymorphism” or similar phrases. The actually detected allele will be apparent in the genomic DNA of interest, but may also be detectable from RNA or protein sequences transcribed or translated from this region.

  Amplification: The use of techniques that increase the number of copies of nucleic acid molecules in a sample. An example of an in vitro amplification method is the polymerase chain reaction (PCR) in which a biological sample obtained from a subject is contacted with a pair of oligonucleotide primers under conditions that allow the oligonucleotide primer to hybridize to nucleic acid molecules in the sample. It is. These primers are extended under appropriate conditions, separated from the template, and reannealed, extended, and separated to amplify the copy number of the nucleic acid molecule. Amplified products can be characterized by techniques such as electrophoresis, restriction enzyme cleavage patterns, oligonucleotide hybridization or ligation, and / or nucleic acid sequencing.

  Other examples of amplification methods include strand displacement amplification methods disclosed in US Pat. No. 5,744,311; transcriptionless isothermal amplification methods disclosed in US Pat. No. 6,033,881; PCT publication Repair chain reaction amplification method disclosed in publication No. WO 90/01069; Ligase chain reaction amplification method disclosed in EP-A-320,308; Gap filling disclosed in US Pat. No. 5,427,930 Ligase chain reaction amplification; and the trademark NASBA-free RNA transcription amplification disclosed in US Pat. No. 6,025,134. The amplification method can be modified, for example, by additional steps or by combining the amplification method with another protocol.

  Array: A molecule, specifically a biopolymer (such as a polypeptide or nucleic acid), or a sample of cells or tissues, arranged in an addressable location on a substrate or in a substrate. A “microarray” is an array that is miniaturized and requires or is assisted by microscopy for evaluation or analysis. These arrays are sometimes called DNA chips, or generally biochips, but more formally called microarrays, and the process of examining an individual's genetic pattern is sometimes called microarray profiling. The manufacturing chemistry and structure of DNA arrays are diverse, typically consisting of 400,000 different features, each holding DNA from a different human gene, but in some cases 780,000 Solid state chemistry may be used to pattern as many individual features.

  An array of molecules ("features") allows a very large number of analyzes to be performed on a sample simultaneously. In certain array examples, for example, one or more molecules (such as oligonucleotide probes) will be present on the array multiple times (such as twice) to provide an internal standard. The number of addressable locations on the array can be, for example, several (such as three) to at least 50, at least 100, at least 200, at least 250, at least 300, at least 500, at least 600, at least It can vary up to 1000, at least 10,000, or more. In a specific example, the array is, for example, an oligonucleotide sequence that is at least 15 nucleotides in length, such as about 15-40 nucleotides in length, such as at least 18 nucleotides in length, at least 21 nucleotides in length, or even Includes nucleic acid molecules of at least 25 nucleotides in length. In one example, the molecule comprises an oligonucleotide attached to the array via its 5 'end or 3' end.

  Within the array, each sample placed in the array is addressable in that it can be determined reliably and consistently within at least two dimensions of the array. The feature application sites on the array can take a variety of forms. For example, the array may be regular (arranged in uniform rows and columns) or irregular. That is, within a regular array, the location of each sample is assigned to that sample when it is applied, and keys may be provided to associate each location with the appropriate target or feature site. Absent. Often the regular array is arranged in a symmetric grid pattern, but the samples can be arranged in another pattern (eg, in a radial, spiral, or regular cluster). Addressable arrays typically have the ability to program a computer to associate a specific address on the array with information about the sample at that location (eg, hybridization or binding data, such as signal intensity) And is computer readable. In some examples of computer readable formats, the individual features in the array are regularly arranged in a Cartesian grid pattern, for example, so that address information can be associated by a computer.

  Also, protein-based arrays where the probe molecule is a protein or a protein, or the target molecule is a protein or a protein, and an array containing nucleic acids to which a protein / peptide is bound, or vice versa, As assumed herein.

  Binding or stable binding: binding between two substances or molecules, such as hybridization of one nucleic acid molecule with another (or itself) and association of an antibody with a peptide. If a sufficient amount of an oligonucleotide molecule forms a base pair or hybridizes with its target nucleic acid molecule, the oligonucleotide molecule binds or stably binds to the target nucleic acid molecule and detects its binding. Is possible. Binding can be detected by any procedure well known to those skilled in the art, such as by physical or functional properties of the target oligonucleotide complex. For example, binding can be functionally detected by determining whether the binding has an observable effect on biosynthetic processes such as gene expression, DNA replication, transcription, translation, and the like.

  Physical methods for detecting the binding of complementary strands of nucleic acid molecules include DNA-degrading enzyme 1 or chemical footprinting, gel shift assay and affinity cleavage assay, Northern blot, dot blot, and light absorption detection However, it is not limited to these. For example, one method involves observing that the light absorption of a solution containing an oligonucleotide (or analog) and a target nucleic acid changes from 220 nm to 300 nm as the temperature is gradually increased. When this oligonucleotide or analog binds to its target, there is a sharp increase in absorption at the characteristic temperature as the oligonucleotide (or analog) and target dissociate from each other or dissolve. In another example, the method includes detecting a signal present on one or both of the complementary strands, eg, a detectable label.

The binding of an oligomer to its target nucleic acid is often characterized by the temperature (T _m ) at which 50% of the oligomer is lost from its target. (T _m) higher, as compared to the complex of lower (T _m), which means that the stronger or more stable complex.

  Complement component 2 (C2): Part of the classical pathway of the complement system. Activation C1 cleaves C2 into C2a and C2b. C2a results in activation of C3. It has been reported that C2 deficiency is associated with certain autoimmune diseases such as systemic lupus erythematosus, Henoch-Schönlein purpura, or polymyositis. C2 is a member of EC 3.4.21.43. This is also known as the classical complement pathway C3 / C5 convertase.

  Complement factor H: also known as β-1H. Serum glycoprotein that regulates the function of the second complement pathway and acts as a cofactor for factor I (C3b inactivator). This regulates the activity of C3 convertases such as C4b2a.

  Complementarity and complementarity: Molecules with complementary nucleic acids bind (hybridize) to each other by strands forming Watson-Crick, Hoogsteen, or reverse Hoogsteen base pairing. When combined, they form a stable duplex or triplex. Stable binding occurs when the oligonucleotide molecule is in a detectably bound state to the target nucleic acid sequence under the required conditions.

  Complementarity is the degree to which a base in one nucleic acid strand is base paired with a base in another nucleic acid strand. Complementarity is conveniently expressed as a percentage, that is, the proportion of nucleotides that form base pairs between the two strands or within a particular region or domain of the two strands. For example, if 10 of 15 oligonucleotides base pair with the target region of the DNA molecule, the oligonucleotide has 66.67% complementarity with the targeted region of DNA. It turns out that.

  In this disclosure, “sufficient complementarity” refers to detectable binding because there is a sufficient number of base pairs between the oligonucleotide molecule and the target nucleic acid sequence (such as the CFH, BF, or C2 sequence). It means reaching. When expressed or measured as a percentage of base pairs formed, the complementation rate that achieves this goal can range from as little as about 50% complementarity to complete (100%) complementarity. In general, sufficient complementarity is at least about 50%, such as at least about 75% complementarity, at least about 90% complementarity, at least about 95% complementarity, at least about 98% complementarity, Or even at least about 100% complementarity.

  A complete treatment of qualitative and quantitative conditions related to defining binding conditions that allow one skilled in the art to design oligonucleotides suitable for use under the desired conditions is described in Beltz et al. (1983). Methods Enzymol 100: 266-285; and Sambrook et al. (Ed.), Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

  DNA (deoxyribonucleic acid): A long polymer that contains the genetic material of most organisms (some viruses have a gene containing RNA that is a ribonucleotide). The repeating unit of the DNA polymer is 4 types of nucleotides, and each of the 4 types of bases (adenine, guanine, cytosine, and thymine) bonded to the deoxyribose sugar to which the phosphate group is attached. Contains one. A triplet of nucleotides, called codons, in the DNA molecule encodes an amino acid of a polypeptide. The term codon is also used for the corresponding (and complementary) sequence of three nucleotides in the mRNA into which the DNA sequence is transcribed.

  Drusen: Deposits that accumulate between the RPE basal layer and the inner colloid layer of Bruch's membrane (eg, van der Schaf et al. (1992) Ophthalmol. 99: 278-86; Spraul et al. (1997) Arch. Ophthalmol. 115: 267. Mullins et al., Histochemical comparison of ocular “drusen” in monkey and human, In M. LaVail, J. Hollyfield, and R. Anderson (Eds.), In Degen. York: See Plenum Press, 1997). Rigid drusen is a small, unique deposit that contains a homogeneous acidophilic material, usually circular or hemispherical, with no sloping edges. Soft drusen is larger, usually not homogeneous, and generally includes inclusions and spherical contours. Some drusen may be calcified. The term “scattered drusen” or “baseline linear deposit” is used to describe the amorphous material that forms a layer between the inner colloid layer of Bruch's membrane and the retinal pigment epithelium (RPE). This material may appear histologically similar to soft drusen, except that it is not raised.

  Factor B (BF): an activator precursor of complement 3 in the second complement activation pathway. Factor b is converted to c3 convertase by factor d. BF is a member of EC 3.4.21.47. Factor B circulates in the blood as a single-chain polypeptide. When the second pathway is activated, it is cleaved by complement factor d to become non-catalytic chain Ba and catalytic subunit Bb. The active subunit Bb is a serine protease that binds to C3b to form the second pathway C3 convertase. BF is also known as the second complement pathway C3 / C5 convertase.

  Genetic predisposition or risk: A subject's susceptibility to a genetic disorder, such as AMD. However, this susceptibility may or may not result in the actual development of the disease.

  Haplotype: The genetic makeup of individual chromosomes. In diploid organisms, the haplotype contains one of the allelic pairs for each site. A haplotype can mean only one locus or the entire genome. A haplotype may also mean a set of single nucleotide polymorphisms (SNPs) that are found to be statistically related on a single chromatid.

  Hybridization: Oligonucleotides and their analogs hybridize between complementary bases by hydrogen bonding, such as Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding. In general, nucleic acids consist of nitrogen-containing bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogen-containing bases form a hydrogen bond between pyrimidine and purine, and this bond between pyrimidine and purine is called “base pairing”. More specifically, A should hydrogen bond with T or U and G should bond with C. “Complementary” means base pairing that occurs between two distinct nucleic acid sequences or between two distinct regions of the same nucleic acid sequence.

  “Specifically hybridizable” and “specifically complementary” are used to produce a stable and specific binding between an oligonucleotide (or analog thereof) and a DNA or RNA target. This term represents a sufficient degree of complementarity. An oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence, which can specifically hybridize. When an oligonucleotide or analog binds to a target DNA or RNA molecule, it interferes with the normal functioning of the target DNA or RNA molecule, and the oligonucleotide or analog is desired for specific binding. An oligonucleotide or analog is specific when there is sufficient complementarity to avoid non-specific binding to sequences other than the target under conditions, for example, physiological conditions in the case of in vivo assays or systems. Can be hybridized. Such binding is called specific hybridization.

Hybridization conditions that produce a particular degree of stringency will vary depending on the nature of the chosen hybridization method and the composition and length of the hybridizing nucleic acid sequences. In general, the stringency of hybridization is determined by the hybridization temperature and the ionic strength of the hybridization buffer (particularly the concentration of Na + and / or Mg ++), although the number of washings also affects stringency. . Calculation method regarding hybridization conditions required to achieve the stringency of a particular degree, Sambrook et al.,, Molecular Cloning (ed.) : A Laboratory Manual, 2 nd ed. , Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11; and Ausubel et al., Short Protocols in Molecular Biology, 4th ed. , John Wiley & Sons, Inc. , 1999.

  For the purposes of this disclosure, “stringent conditions” includes conditions in which hybridization occurs only if there is less than 25% mismatch between the hybridizing molecule and the target sequence. “Stringent conditions” may be categorized into a particular level of stringency for more precise definition. That is, in this specification, “slow stringency” conditions are conditions in which molecules with sequence mismatches greater than 25% do not hybridize, and “moderate stringency” conditions are sequences greater than 15%. Mismatched molecules are conditions that do not hybridize, and “high stringency” conditions are those where molecules with sequence mismatch greater than 20% do not hybridize. “Extremely high stringency” conditions are conditions in which molecules with sequence mismatches greater than 10% do not hybridize.

  The following are examples of a series of hybridization conditions and are not limiting.

Extremely high stringency (detects sequences with 90% identity)
Hybridization: 5 × SSC, washed twice for 16 hours at 65 ° C .: 2 × SSC, washed twice for 15 minutes at room temperature (RT): 0.5 × SSC, washed for 20 minutes at 65 ° C. High stringency (Detect sequences with more than 80% identity)
Hybridization: 5 × -6 × SSC, washed twice at 65 ° C.-70 ° C. for 16-20 hours, 2 times: 2 × SSC, washed twice at RT for 5-20 minutes: 1 × SSC, 55 ° C.-70 ° C. Low stringency for 30 minutes each (detects sequences with 50% or more identity)
Hybridization: 6 × SSC, at RT-55 ° C. for 16-20 hours at least twice Washing: 2 × -3 × SSC, RT-55 ° C. for 20-30 minutes each Isolated: “Isolated "A biological component (such as a nucleic acid molecule, protein, or organelle) is another biological component within the cell of the organism in which it naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles. From or purified from. “Isolated” nucleic acid molecules and proteins include nucleic acid molecules and proteins purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acid molecules and proteins.

  Linkage disequilibrium (LD): A non-random association of alleles at two or more loci that are not necessarily on the same chromosome. LD is present in a population at a frequency that is higher or lower than expected when some combination of alleles or genetic markers is based on their frequency and haplotypes are randomly formed from alleles It represents the state of being. The expected frequency of two alleles that are inherited independently is the frequency of the first allele multiplied by the frequency of the second allele. Alleles that coexist at the expected frequency are said to be in linkage equilibrium.

  Locus: The location of a gene (or other important sequence) on a chromosome.

  Mutation: A change in the DNA sequence within a gene or chromosome. In some instances, a mutation changes a characteristic or trait (phenotype), but not necessarily. Types of mutations include base substitution point mutations (eg, transition or transversion), deletions, and insertions. A missense mutation is a mutation that introduces another amino acid into the sequence of the encoded protein, and a nonsense mutation is a mutation that introduces a new stop codon. In the case of an insertion or deletion, the mutation may be in-frame (does not change the frame of the entire sequence) or may be a frameshift mutation, which can lead to misreading of many codons (And also often results in abnormal termination of the encoded product due to the presence of a stop codon in another frame).

  This term specifically includes mutations caused by somatic mutations, for example, mutations that are found only in diseased cells in a particular individual but not constitutionally. Examples of such somatically acquired mutations include point mutations that often result in altered function of various genes involved in the development of cancer. The term also includes alterations in DNA that are constitutionally present, alter the function of the encoded protein in a way that is readily apparent, and that may be inherited by children of affected individuals. In this regard, the term overlaps with the “polymorphism” defined below, but generally refers to a subset of constitutional changes.

  Nucleic acid molecule: A polymeric form of nucleotide, including RNA, cDNA, genomic DNA, and both the sense and antisense strands of these synthetic and mixed polymers. Nucleotide means ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide. As used herein, “nucleic acid molecule” is synonymous with “nucleic acid” and “polynucleotide”. A nucleic acid molecule is usually at least 10 bases in length, unless otherwise noted. The term includes single and double stranded DNA. A polynucleotide can comprise one or both of natural and modified nucleotides linked by natural and / or non-natural nucleotide bonds.

  Nucleotides: bases linked to sugars, such as pyrimidines, purines or synthetic analogs thereof, or monomers containing bases linked to amino acids as in peptide nucleic acids (PNA), but are not limited thereto. A nucleotide is a single monomer in a polynucleotide. Nucleotide sequence means the sequence of bases in a polynucleotide.

  Oligonucleotide: A nucleic acid molecule generally consisting of 300 bases or less in length. The term often refers to single-stranded deoxyribonucleotides, but also refers to, inter alia, single- or double-stranded ribonucleotides, RNA: DNA hybrids, and double-stranded DNA. The term “oligonucleotide” also includes oligonucleosides (ie, oligonucleotides without phosphate) and other organic base polymers. In some examples, the oligonucleotide is about 10 to about 90 bases in length, eg, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases in length. Other oligonucleotides are about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 bases, about 65 bases, about 70 bases, about 75 bases, or about 80 bases in length. It is. Oligonucleotides may be single stranded, for example, for use as a probe or primer, and double stranded, for example, for use in constructing a mutant gene. The oligonucleotide may be either a sense oligonucleotide or an antisense oligonucleotide. Oligonucleotides can be modified as described above for nucleic acid molecules. Oligonucleotides can be obtained from existing nucleic acid-derived sources (eg, genomic DNA or cDNA), but can also be synthesized (eg, generated by oligonucleotide synthesis in the laboratory or in vitro).

  Polymorphism: A mutation in a gene sequence. Polymorphisms are mutations (DNA sequence differences) found between individuals or between different ethnicities and between geographical locations, producing functionally equivalent gene products even with different sequences It may be a mutation. The term also refers to sequence variants that can result in a gene product that is not functionally equivalent. Polymorphisms also include mutations that can be classified as alleles and / or mutations that can give rise to gene products that may have altered function. Polymorphisms also do not give rise to gene products, or produce inactive gene products, or active gene products at abnormal rates, in inappropriate tissues, or in response to inappropriate stimuli Mutations that can be classified as alleles and / or mutations. In addition, the term is used interchangeably with allele, where appropriate.

  A polymorphism may be referred to, for example, at a nucleotide position where a mutation is present, in an amino acid sequence change caused by the nucleotide mutation, or in some other characteristic change in a nucleic acid molecule or protein associated with the mutation.

  Probes and primers: Probes include identifiable, isolated nucleic acids that recognize target nucleic acid sequences. A probe includes a nucleic acid that is attached to an addressable location, a detectable label, or other reporter molecule that hybridizes to a target sequence. Common labels include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in selecting labels suitable for various purposes are described, for example, in Sambrook et al. (Ed.), Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel et al., Short Protocols in Molecular Biology, 4th ed. , John Wiley & Sons, Inc. , 1999.

  A primer is a short nucleic acid molecule, such as a DNA oligonucleotide having a length of 10 nucleotides or more that hybridizes to a contiguous complementary nucleotide or sequence to be amplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and target DNA strand, and then the primer is extended along the target DNA strand by a DNA polymerase enzyme Can do. As described later, the primer pair can be used for amplification of a nucleic acid sequence by, for example, a PCR method or a nucleic acid amplification method known in the art.

  Methods for preparing and using nucleic acid probes and primers are described, for example, in Sambrook et al. (Ed.), Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Short Protocols in Molecular Biology, 4th ed. , John Wiley & Sons, Inc. , 1999; and Innis et al. PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. , San Diego, CA, 1990. Amplification primer pairs can be obtained from known sequences using, for example, computer programs for that purpose, such as Primer (Version 0.5, Copyright 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One skilled in the art will appreciate that the specificity of a particular probe or primer increases as its length increases. Thus, to obtain greater specificity, probes or primers can be selected that contain at least 20, 25, 30, 35, 40, 45, 50, or more contiguous nucleotides of the target nucleotide sequence.

  Sample: A sample obtained from a human or non-human mammalian subject. As used herein, biological samples are cells, tissues, and body fluids such as blood; blood derivatives and fractions (such as serum or plasma); extracted bile; biopsy tissue or surgically isolated tissue, eg, fixed Untipped, frozen, formalin-fixed, and / or paraffin-embedded tissue; tears; milk; skin chips; surface wash; urine; sputum; cerebrospinal fluid; prostate fluid; pus; Includes, but is not limited to, all samples useful for genetic analysis within a subject, including aspirate; BAL; saliva; cervical wipes; vaginal wipes;

  Single nucleotide polymorphism or SNP: DNA sequence variation that occurs when homologous companions are single nucleotides in the genome; adenine (A), thymine (T), cytosine (C) or guanine (G) are different. As used herein, the term “single nucleotide polymorphism” (or SNP) includes mutations and polymorphisms. SNPs may be contained within a gene coding sequence (CDS) or between genes (intergenic region). SNPs inside the CDS change codons and may or may not change amino acids in the protein sequence. The former may constitute different alleles. The latter is called a silent mutation and generally occurs at the third position of the codon (called the fluctuating position).

Subject: A human or non-human mammal (such as a veterinary subject).
Methods of identifying subjects at increased risk of AMD Provided are methods for identifying subjects at increased risk of developing age-related macular degeneration (AMD). These methods include analyzing a subject's factor B (BF) and / or complement component 2 (C2) gene and determining whether the subject has at least one protective polymorphism. . However, the protective polymorphisms are from a) R32Q (rs641153) in BF; b) L9H (rs4151667) in BF; c) IVS10 (rs547154) in C2; and d) E318D (rs93332739) in C2. Selected from the group consisting of If the subject does not have at least one protective polymorphism, the subject is at increased risk of developing AMD. The method may further comprise analyzing the subject CFH gene, or any other desired gene. As described herein, the delTT polymorphism within the CFH gene has been identified as protective for AMD.

  The method also analyzes the subject's genotype at either the BF or C2 locus, and the CFH locus, and the subject is heterozygous for a) the R32Q (rs641153) polymorphism in BF; b A) heterozygous for L9H (rs4151667) polymorphism in BF; c) heterozygous for IVS10 (rs547154) polymorphism in C2; and d) heterozygous for E318D (rs93332739) polymorphism in C2; e) in CFH Determine if it has at least one protective genotype selected from the group consisting of homozygous for the delTT polymorphism; and f) homozygous for the R150R (rs1048709) polymorphism in BF and homozygous for Y402 in CFH To include. However, if the subject does not have at least one protective genotype, the subject's risk of developing AMD is increased. Alternatively, the method may comprise analyzing the genotypes of interest at the BF and C2 loci as well as the CFH locus. In addition, the method provided herein can be used to determine whether a subject has a reduced risk of developing AMD by determining whether the subject has at least one of the polymorphisms or genotypes shown above. It is also useful for identification.

  Analysis of a subject's genetic material for the presence or absence of a particular polymorphism is performed by obtaining a sample from the subject. The sample may be from any part of the subject's body from which DNA or RNA can be isolated. Analysis may be performed on the protein isolated from the sample. Examples of such samples will be described in more detail later. The subject may have been diagnosed with AMD, such as early AMD, choroidal neovascularization, or geographic atrophy. Subjects are AMD symptoms such as drusen, pigment changes, exudative changes such as bleeding, hard vitiligo, or subretinal / sub-RPE / intraretinal fluid, vision loss, blurred vision, astigmatism (metastasis), center It may have a dark spot or color blindness. Alternatively, the subject may have never been diagnosed with AMD, but may be in a high risk group based on family history, age, race, or lifestyle options. These lifestyle options include smoking, sun exposure (especially blue light), high blood pressure, cardiovascular risk factors such as high cholesterol and obesity, high fat intake, and oxidative stress. It is not limited. Subjects at risk of developing AMD also include those who are heterozygous or homozygous for the risk haplotype Y402H in the CFH gene.

  Techniques for determining the presence or absence of a particular polymorphism or genotype of interest are well known in the art. Examples of these methods are described below, but are not intended to be limited to the specific methods used. Further, analyzing a BF gene, C2 gene, or CFH gene of interest for a particular polymorphism disclosed herein can also detect mutations that result in amino acid variations found in that polymorphism. Shall be included. For example, the L9H polymorphism in BF changes the nucleotide codon for the 9th amino acid from CTC to CAC to produce histidine instead of leucine. This mutation can also be identified by the fact that the CAT nucleotide codon is CAT. The E318D polymorphism in C2 changes the nucleotide codon for the amino acid at position 318 from GAG to GAC, producing aspartic acid instead of glutamic acid. This mutation can also be identified by the nucleotide codon being GAT. The R150R polymorphism in BF changes the nucleotide codon for the amino acid at position 150 from CGG to CGA. This mutation does not change the encoded amino acid (arginine). Arginine can also be encoded by CGT or CGC. In addition, arginine can be encoded by AGA or AGG. The Y402H polymorphism in CFH changes the nucleotide codon for the amino acid at position 402 from TAT to CAT, generating histidine instead of tyrosine. This mutation can also be identified by the nucleotide codon being CAC. Any of these nucleotide codons, or other nucleotide codons that can be identified by one skilled in the art, can also be detected in a subject.

By the methods of the present invention, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 45% of subjects who develop AMD 50%, about 55%, about 60%, about 65%, about 70% can be identified.
AMD Prophylactic Therapy The present disclosure also provides a method of avoiding or reducing the occurrence of AMD in a subject determined to have a genetic predisposition to develop AMD. For example, when using the methods described above, a BF gene, C2 gene, and / or CFH gene has a mutation or protective polymorphism in a subject at risk of developing AMD based on any of the aforementioned risk factors. If not identified, lifestyle choices can be imposed on the subject to avoid or reduce the occurrence of AMD or to delay the onset of AMD. For example, the subject may quit smoking; change the diet to include less fat intake; increase intake of antioxidants such as vitamins C and E, β-carotene, and zinc; A prophylactic amount of a drug that delays the onset may be taken. Treatment for such individuals may include vaccines against certain pathogens, or antibiotics, or antiviral drugs, or antifungal drugs. The therapy may also include anti-inflammatory drugs or complement inhibitors. In some examples, the selected therapy is specific and individually adapted to the subject based on analysis of the subject's genetic profile.
Known polymorphism detection methods Known polymorphism detection methods include restriction fragment length polymorphism (RFLP), single-stranded DNA conformation polymorphism (SSCP), mapping, nucleic acid sequencing, high Hybridization method, fluorescence in situ hybridization (FISH) method, PFGE analysis method, RNase protection assay method, allele-specific oligonucleotide (ASO) method, dot blot analysis method, allele-specific PCR amplification (ARMS) method , Oligonucleotide ligation assay (OLA) method, and PCR-SSCP method, but are not limited thereto. Recently developed mass spectrometry methods (such as matrix-assisted laser desorption / ionization (MALDI) or time-of-flight MALDI (MALDI-TOF) methods) and DNA microchip technology for detecting mutations are also useful. For example, Human Molecular Genetics2. Tom Strachan and Andrew, Read. New York: John Wiley & Sons Inc. , 1999, chapters 6 and 17.

  These techniques may include amplifying the nucleic acid prior to analysis. Amplification techniques are well known to those skilled in the art and will be described later.

  When a polymorphism causes a nucleotide mutation that creates or destroys a recognition site for a restriction enzyme, the restriction enzyme can be used to identify the polymorphism. Polymorphic alleles can be distinguished by PCR amplification of the entire polymorphic site followed by degradation of the PCR product with associated restriction enzymes. Various products can be detected using size fractionation methods such as gel electrophoresis. Alternatively, the restriction fragment length polymorphism (RFLP) method can be used. If the polymorphism does not result in a restriction site difference, the difference between alleles can be detected by restriction site PCR created by amplification. In this method, primers are designed from sequences that are immediately adjacent to, but do not include, a restriction site. This primer is carefully designed to have a single base mismatch at an insignificant position that does not prevent hybridization and amplification of both polymorphic sequences. This nucleotide mismatch, together with the sequence at the polymorphic site, creates a restriction site that is not present on one of the alleles.

  The single-strand conformation polymorphism (SSCP) mapping method detects a band that has shifted differentially due to a difference in the intramolecular base pairing of a single strand caused by a sequence change. Single-stranded DNA molecules that differ by only one base often exhibit different electrophoretic mobilities in non-denaturing gels. The difference in mobility between normal DNA and mutant DNA is revealed by hybridization using a labeled probe. In particular, if the size of the DNA fragment is greater than about 500 bp, not all sequence changes can be detected by this method, but can be optimized to detect most DNA sequence variations. Although low detection sensitivity is inconvenient, SSCP allows for increased throughput, making this an attractive alternative to direct sequencing for mutation detection on a research basis. Yes. Therefore, the exact nature of the DNA sequence variation is measured by sequencing fragments with different mobility on an SSCP gel.

  Sequence variations can be detected by direct DNA sequencing by either manual sequencing or automatic fluorescent sequencing.

  Taq polymerase can be used to detect specific alleles (Holland et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7276-80; Lee et al. (1999) J Mol.Biol.285: 73-83). This is based on the fact that Taq polymerase does not have a proofreading 3 '→ 5' exonuclease activity but has a 5 '→ 3' exonuclease activity. This assay specifically binds to two conventional PCR primers that are specific to the target sequence (forward and reverse) and a site on the target sequence downstream of the forward primer binding site. Using a designed third primer. The third primer is usually labeled with two fluorophores: a reporter dye at the 5 'end and a quencher dye having an emission wavelength different from that of the reporter dye at the 3' end. The third primer also carries a protecting group on the 3 'terminal nucleotide so that new DNA synthesis cannot be initiated by itself. In the course of the PCR reaction, Taq DNA polymerase synthesizes a new DNA strand initiated by the forward primer, and as its enzyme approaches the third primer, its 5 ′ → 3 ′ exonuclease activity causes The 3 primers are gradually degraded from their 5 ′ ends. The net result is that the nascent DNA strand extends beyond the third primer binding site and the reporter dye and quencher dye can no longer bind to the same molecule. When the reporter dye is no longer present in the vicinity of the quencher dye, an increase in the reporter's emission intensity results, which can be detected.

  A polymorphism can be identified using one or more hybridization probes designed to hybridize to a particular polymorphism within the desired gene. Probes used in the hybridization detection method must be labeled in some way so that successful hybridization can be detected. This can be done by in vitro methods such as nick translation, which replaces nucleotides in the probe with radiolabeled nucleotides, or by random primer extension methods where unlabeled molecules act as templates to synthesize labeled copies. It can be carried out. Other standard methods for labeling probes to detect hybridization are known to those skilled in the art.

  For DNA fragments up to about 2 kb in length, a single base change can be detected by chemically cleaving at a mismatched base in the mutant-normal heteroduplex. For example, one end of a DNA strand not containing the desired polymorphism is radiolabeled and then hybridized with one strand of the target DNA strand. When the resulting heteroduplex DNA is treated with hydroxylamine or osmium tetroxide, they modify C, or C and T, respectively, in the unpaired single stranded region, and the modified backbone is cleaved by piperidine It becomes easy to be done. The shortened labeled fragment is detected by gel electrophoresis and autoradiography while comparing with the DNA not containing the desired polymorphism.

  A mismatch is a hybridized nucleic acid duplex where the two strands are not 100% complementary. The overall lack of homology may be due to deletions, insertions, inversions, or substitutions. Mismatch detection can be used to detect point mutations within a gene or its mRNA product. These techniques are less sensitive than sequencing methods, but are easy to implement on a large number of samples. An example of a mismatch cleavage technique is the RNase protection method. This method involves the use of a labeled riboprobe that is complementary to one mutation of the polymorphism being detected (usually a polymorphism not associated with protection from AMD). RNA that is an enzyme capable of detecting several mismatches in a double-stranded RNA structure after annealing (hybridizing) the riboprobe and either mRNA or DNA isolated from a subject together Decompose with degrading enzyme A. When a mismatch is detected by RNase A, it is cleaved at the mismatch site. Thus, when the annealed RNA preparation is separated on an electrophoresis gel matrix, if the mismatch is detected by RNase A and cleaved, full-length double-stranded RNA corresponding to the riboprobe, and mRNA Or an RNA product shorter than DNA should be seen. The riboprobe need not be the full length of the mRNA or gene, but can be either segment. Alternatively, the mismatch can be detected by a change in the electrophoretic mobility of the mismatched duplex compared to the matched duplex.

  The DNA sequence of each gene of BF, C2 or CFH amplified using the PCR method can also be screened using an allele-specific probe or oligonucleotide (ASO). These probes are nucleic acid oligomers, each of which contains a region of a gene sequence with a known mutation or polymorphism. For example, an oligomer may be about 30 nucleotides in length and correspond to a portion of the sequence of the BF gene, C2 gene, or CFH gene. A series of such allele-specific probes can be used to screen PCR amplification products to identify the presence of one or more polymorphisms described herein. Hybridization of allele-specific probes to amplified BF, C2 or CFH sequences can be performed, for example, on a nylon filter. Alternatively, the reverse dot blot method may be used. For example, screening for one or more polymorphisms is a series of ASOs specific for each polymorphic allele on a single membrane that will hybridize to labeled PCR amplified test DNA. This can be done with a series of spotted ASOs. These assays range from a small number of manually spotted arrays to very large ASO arrays on “gene chips” with the potential to detect multiple polymorphisms. Hybridizing to a specific probe under high stringency hybridization conditions indicates that the same polymorphism in the allele-specific probe is present in the tissue. Such techniques can utilize probes that are labeled with gold nanoparticles to give a visible color result (Elghanian et al. (1997) Science 277: 1078-81).

  Allele-specific PCR amplification is based on a method called the amplification refractory mutation system (ARMS) (Newton et al. (1989) Nucleic Acids Res. 17: 2503-16). In this method, oligonucleotides with mismatched 3'-residues do not function as PCR primers under appropriate conditions. Paired PCR reactions are performed using two types of primers, one common primer and the other two specific primers that are present in two slightly different forms. These allele-specific primers are designed to be identical to the sequences of the two alleles over a region that precedes and continues to the mutant nucleotide itself. Thus, no amplification product is observed unless a specific polymorphism or mutation is present. In general, separate control primers are used to amplify unrelated sequences. The position of the common primer can be designed to produce different sized products for different polymorphisms, so that the PCR product from the multiplex reaction forms a ladder on the gel. The polymorphic specific primer can be labeled with a variety of fluorescent labels or other labels, or a 5 'extension of various sizes may be added. This method can be adapted for use in real-time PCR.

  In the oligonucleotide ligation assay (OLA), two oligonucleotides are designed to hybridize to flanking sequences within the target. The site where these bind is the polymorphic site. Only when these two oligonucleotides are completely hybridized, DNA ligase will link the two oligonucleotides (Nickerson et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8923-7). This assay can use a variety of formats, such as ELISA analysis or fluorescence sequencing.

  Nucleic acid analysis technology using microchip technology can also be used. With this technique, it is possible to create thousands of individual oligonucleotide probes in an array on a silicon chip. The nucleic acid to be analyzed is fluorescently labeled and hybridizes with these probes on the chip. These nucleic acid microchips can be used to examine nucleic acid-protein interactions. This technique can be used to determine the presence of a mutation and to further sequence the nucleic acid under analysis or to measure the expression level of the gene of interest. This method is a method of processing thousands of probes that may be thousands at the same time, and can greatly increase the analysis speed.

  Changes in BF, C2, or CFH mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification, and RNase protection. A decrease in mRNA expression indicates that the wild type gene has changed. If a particular allele produces a protein with an amino acid mutation, the allele detection method can be based on the protein. For example, epitopes specific for amino acid mutations can be detected by monoclonal antibodies. Alternatively, tissues can be screened using monoclonal antibodies that are immunoreactive with BF, C2, or CFH. If no alloantigen is present, it will indicate that there is a mutation. It is also possible to detect a mutant gene product using an antibody specific for the mutant allele product. Such immunoassays can also be performed in any convenient format known in the art. These include Western blots, immunohistochemical assays, and ELISA assays. Any means for detecting the altered protein can be used to detect alteration of the wild-type BF gene, C2 gene, or CFH gene. Functional analysis methods such as protein binding measurement methods can be used. In addition, assays that detect the biochemical function of BF, C2, or CFH can be used. If a mutant BF, C2, or CFH gene product is found, it indicates that the wild-type BF, C2, or CFH gene has been altered.

Amplification of nucleic acid molecules A nucleic acid sample obtained from a subject may be amplified from a clinical sample prior to detection. In one embodiment, the DNA sequence is amplified. In another embodiment, the RNA sequence is amplified.

  Any nucleic acid amplification method can be used. In one specific, non-limiting example, the polymerase chain reaction (PCR) method is used to amplify nucleic acid sequences associated with AMD. Examples of other methods include RT-PCR and transcription-mediated amplification (TMA), cloning, allele-specific polymerase chain reaction (PASA), ligase chain reaction, and nested polymerase chain reaction. It is not limited to these.

  The primer pair can be used for an amplification reaction. One or both primers can be labeled, for example, with a detectable radiolabel, fluorophore, or biotin molecule. The primer pair can include an upstream primer (binding to 5 'of the downstream primer) and a downstream primer (binding to 3' of the upstream primer). The primer pair used in the amplification reaction may be a selection primer that allows amplification of nucleic acids related to AMD.

  Another primer pair can be included in the amplification reaction as an internal control. For example, these primers can be used to amplify “housekeeping” nucleic acid molecules and help confirm that proper amplification has occurred. In another example, a target nucleic acid molecule that includes a primer hybridization site can be constructed and placed in an amplification reactor. One skilled in the art can readily identify a primer pair that serves as an internal control primer.

  Amplification products can be analyzed in various ways, including size analysis, size analysis after digestion with restriction enzymes, detection of specific labeled oligonucleotide primers in reaction products, allele-specific oligonucleotide (ASO) hybridization, sequencing, and hybridization. Can be assayed.

Detection assays based on PCR methods involve multiplex amplification of multiple polymorphisms simultaneously. For example, it is well known in the art to select PCR primers to create PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, because they are differentially labeled, various polymorphisms can be amplified with primers that can detect each. Other techniques are known in the art that allow multiple analyzes of multiple polymorphisms. A fragment of a gene can be amplified to make a copy, and it can be determined whether the copy of the fragment contains a particular protective polymorphism or genotype.
Protective Protein Immunodetection Methods In one embodiment of the invention, a protein assay is performed to characterize a polymorphism in a C2 or BF gene of interest, eg, to detect or identify a protective protein. Methods that can be adapted to detect mutant proteins are well known in the art, analytical biochemical methods such as electrophoresis (including capillary electrophoresis and two-dimensional electrophoresis), fast Liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, chromatographic methods such as mass spectrometry, liquid precipitin reaction or gel preprecin reaction, immunodiffusion method (one and two dimensions), immunity There are immunological methods such as electrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and Western blot.

  For example, several established immunological binding assay formats suitable for practicing the present invention are known (eg, Harlow, E .; Lane, D. Antibodies: A laboratory manual. Cold Spring Harbor, N.Y .: Cold Spring Harbor Laboratory; 1988; and Ausubel et al. (2004) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY). This assay may be, for example, competitive or non-competitive. In general, immunological binding assays (ie immunoassays) specifically bind to an analyte and often utilize “supplementing agents” to immobilize it. In one embodiment, the supplement is a moiety that specifically binds to a mutant C2 or BF polypeptide or subsequence. The binding protein can be detected using, for example, a detectably labeled anti-C2 / BF antibody. In one embodiment, at least one of the antibodies is specific for a variant (eg, does not bind to a wild type C2 polypeptide or a BF polypeptide).

  Thus, in one aspect, the method obtains a sample (eg, blood, serum, plasma, or urine) from a subject and binds the sample to distinguishing between protective and non-protective C2 or BF. Contacting the agent and detecting that a complex has formed between the binding agent and, if present, unprotected C2 or BF. It will be appreciated that an antibody panel can be used to detect protective proteins in a patient sample.

  The invention also provides an antibody that specifically binds to a protective C2 protein or DF protein, but does not specifically bind to a wild-type polypeptide (ie, a C2 protein or BF protein that is not associated with defense). These antibodies bind to epitopes that exist only in the protective form. For example, certain antibodies do not bind to wild-type BF (Genbank accession number. NM — 001710; encoded by AAB67977) or C2 (Genbank accession number. NM — 000063; encoded by NP — 000054) as described above. , Binds to a variant of BF or C2 (ie, a protein having one of the polymorphisms described herein as being protective against AMD). For example, the antibody can recognize a BF protein having glutamine at position 32 or having histidine at position 9 or C2 having aspartic acid at position 318.

The antibody may be polyclonal or monoclonal and is made according to standard protocols. Antibodies can be made by injecting a protective protein or fragment thereof into a suitable mammal. Screen monoclonal antibodies according to standard protocols (Koehler and Milstein 1975, Nature 256: 495; Dower et al., WO 91/17271, and McCafferty et al., WO 92/01047; and Vauhan et al., 1996, Nature Biotechnology, 14: 309; References below). Monoclonal antibodies can be assayed for specific immune activity using methods well known in the art, using protective polypeptides rather than the corresponding wild-type polypeptides. For methods such as antibody screening methods and subtraction methods, see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Current Protocols in Immunology. , Inclusion supplements through 2005); Goding, Monoclonal Antibodies, Principles and Practice (2d ed.) Academic Press, New York et al. (1986); cular cloning of rare antiviral antibody specificities from phage display libraries "Res Virol. 149 (5): 327-30; Ames et al., 1994, Isolation of neutralizing anti-C5a monoclonal antigens, from a filamentous phage display Fab display library. J Immunol. 152 (9): 4572-81; Shinohara et al., 2002, Isolation of monoclonal antibodies recognizing rare and dominant epithelium in plant vascular bills bis See J Immunol Methods 264 (1-2): 187-94. Immunization or screening may be against full-length protective proteins, or (often more conveniently) against peptides or polypeptide fragments containing epitopes known to differ between mutant and wild type It may be. The antibody may be a tetramer comprising two light chains and two heavy chains, as separate heavy chains, light chains, Fab, Fab ′, F (ab ′) ₂ , and Fv, or as heavy chains and It can be expressed as a single chain antibody in which the variable domains of the light chain are linked via a spacer.

Amplification of nucleic acid molecules A nucleic acid sample obtained from a subject may be amplified from a clinical sample prior to detection. In one embodiment, the DNA sequence is amplified. In another embodiment, the RNA sequence is amplified.

  Any nucleic acid amplification method can be used. In one specific, non-limiting example, the polymerase chain reaction (PCR) method is used to amplify nucleic acid sequences associated with AMD. Examples of other methods include RT-PCR and transcription-mediated amplification (TMA), cloning, allele-specific polymerase chain reaction (PASA), ligase chain reaction, and nested polymerase chain reaction. It is not limited to these.

  The primer pair can be used for an amplification reaction. One or both primers can be labeled, for example, with a detectable radiolabel, fluorophore, or biotin molecule. The primer pair can include an upstream primer (binding to 5 'of the downstream primer) and a downstream primer (binding to 3' of the upstream primer). The primer pair used in the amplification reaction may be a selection primer that allows amplification of nucleic acids related to AMD.

  Another primer pair can be included in the amplification reaction as an internal control. For example, these primers can be used to amplify “housekeeping” nucleic acid molecules and help confirm that proper amplification has occurred. In another example, a target nucleic acid molecule that includes a primer hybridization site can be constructed and placed in an amplification reactor. One skilled in the art can readily identify a primer pair that serves as an internal control primer.

  Amplification products can be analyzed in various ways, including size analysis, size analysis after digestion with restriction enzymes, detection of specific labeled oligonucleotide primers in reaction products, allele-specific oligonucleotide (ASO) hybridization, sequencing, and hybridization. Can be assayed.

Detection assays based on PCR methods involve multiplex amplification of multiple polymorphisms simultaneously. For example, it is well known in the art to select PCR primers to create PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, because they are differentially labeled, various polymorphisms can be amplified with primers that can detect each. Other techniques are known in the art that allow multiple analyzes of multiple polymorphisms. A fragment of a gene can be amplified to make a copy, and it can be determined whether the copy of the fragment contains a particular protective polymorphism or genotype.
Complement factor H (CFH)
The CFH gene is located in a region that is repetitively associated with AMD on the Iq chromosome in family-based studies. Recently, three independent studies have changed the tyrosine to histidine within the complement factor H gene (Y402H), a polymorphism of T → C substitution at nucleotide position 1277 in exon 9 has become a susceptibility to AMD. It has been shown to be substantially involved (Klein et al. (2005) Science 308: 385-389; Haines et al. (2005) Science. 308: 41-9-421; Edwards et al. (2005) Science. 308: 421) 424). These studies reported AMD odds ratios of 3.3 to 4.6 for carriers of the C allele and 3.3 to 7.4 for CC homozygotes. Two other studies subsequently confirmed this linkage (Zareparsi et al. (2005) Am. J. Hum. Genet. 77: 149-153; Hageman et al. (2005) Proc. Natl. Acad. Sci. U.S.). S.A. 102: 7227-7232). In one study, seven other common SNPs were found to be associated with AMD in addition to the Y402H polymorphism (Hageman et al. (2005) Proc. Natl. Acad. Sci. USA 102. : 7227-7232).

  Pair linkage analysis revealed that the seven polymorphisms were in linkage disequilibrium, and one common risk haplotype with a set of these polymorphisms was detected in 50% of patients versus 29% of controls [OR = 2.46, 95% CI (1.95-3.11)]. This haplotype homozygote was found in 24.2% of cases and 8.3% of controls. Two common protective haplotypes were also found in 34% of controls and 18% of cases [OR = 0.48, 95% CI (0.33-0.69)] and [OR = 0.54, 95% CI (0.33-0.69)].

Factor B and complement component 2
Activation of the second pathway is initiated by cleaving factor B bound to C3b (BF) catalyzed by factor D, resulting in the formation of a C3Bb complex (C3 convertase). This complex is stabilized by the regulatory protein properdin, while its dissociation is facilitated by regulatory proteins such as CFH. BF and C2 are paralogous genes that are located only 500 bp apart on human chromosome 6p21. C2 functions within the classical complement pathway. These two genes, together with the genes encoding complement components 4A (C4A) and 4B (C4B), between HLA-B and HLA-DR / DQ within the major histocompatibility complex (MHC) class III region It contains a “comprotype” (complement haplotype) occupying about 100-120 kb.

Clinical samples Suitable for use with the present disclosure in determining a subject's predisposition to genetic predisposition to AMD are blood or blood fractions (such as serum or plasma), gargles or oral cavity scrapings, Conventional clinical samples including, but not limited to, chorionic biopsy samples, semen, Guthrie card, intraocular fluid, sputum, lymph fluid, urine, and tissue. Most simply, blood can be collected and DNA (or RNA) extracted from blood cells. Wild type BF, C2, and / or CFH allelic changes can be detected by any of the means described herein, for example, point mutations or deletions.

  Techniques for obtaining such samples are well known in the art (see, eg, Schluger et al. (1992) J. Exp. Med. 176: 1327-33 for collection of serum samples). Serum or other blood fractions can be prepared by conventional methods. For example, DNA used in the amplification reaction can be extracted using about 200 μL of serum.

  Once a sample is obtained, it can be used directly, concentrated (eg, by centrifugation or filtration), purified, or a combination thereof and an amplification reaction can be performed. For example, DNA preparation can be rapidly performed using a commercially available kit (the InstaGene Matrix, BioRad, Hercules, CA; the NucliSens isolation kit, Organon Teknika, Netherlands, etc.). In one example, this DNA preparation method provides a nucleotide preparation that is readily available for nucleic acid amplification and can be easily performed.

Microarrays In certain instances, methods disclosed in the present specification use polymorphisms in the BF gene, C2 gene, and / or CFH gene. Such arrays can include nucleic acid molecules. In one example, the array includes nucleic acid oligonucleotide probes that can hybridize to polymorphic BF, C2, and / or CFH gene sequences, such as the polymorphisms disclosed herein. Certain of such arrays (as well as the methods disclosed herein) are associated with the risk of developing AMD or other polymorphisms associated with protection therefrom, and one or more housekeeping Other sequences can be included, such as one or more probes that recognize the gene.

  The array termed “AMD detection array” herein is used to determine the genetic susceptibility of a subject to develop AMD. In one example, a set of oligonucleotide probes, for example, for use in detecting polymorphisms in a BF gene, a C2 gene, and / or a CFH gene, which is a gene that amplifies a nucleic acid sequence obtained from a subject. It adheres to the surface of the solid support. Furthermore, if the nucleic acid sequence for internal control is amplified in the amplification reaction (see above), an oligonucleotide probe for detecting the presence of this amplified nucleic acid molecule can be added.

  Nucleotide probes bound to the array can specifically bind to sequences amplified in amplification reactions (such as under high stringency conditions). Oligonucleotides comprising at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of the BF, C2, and / or CFH genes can also be used.

  The method and apparatus according to the present disclosure takes advantage of the fact that under appropriate conditions, an oligonucleotide forms a base-paired duplex with a nucleic acid molecule having a complementary base sequence. This duplex stability depends on several factors, such as the length of the oligonucleotide, the base composition, and the composition of the solution that causes hybridization. The effect of base composition on duplex stability can be reduced by performing hybridization in certain solutions, for example, in the presence of high concentrations of tertiary or quaternary amines.

  Double-stranded thermal stability also depends on the degree of sequence similarity between the sequences. By performing hybridization at a temperature close to the expected Tm for the type of duplex expected to form between the target sequence and the oligonucleotides bound to the array, the mismatched duplex is The rate of formation can be substantially reduced.

  The length of each oligonucleotide sequence used in the array can be selected to optimize the binding of the target BF, C2, and / or CFH nucleic acid sequences. Under specific screening conditions, the optimal length for use with a particular BF, C2, and / or CFH nucleic acid sequence can be determined empirically. Thus, the length of each individual element of the set of oligonucleotide sequences contained in the array can be optimized for screening. In one example, the oligonucleotide probe is about 20 to about 35 nucleotides in length, or about 25 to about 40 nucleotides in length.

  The sequence of oligonucleotide probes forming the array can be bound directly to the support, for example via the 5 'or 3' end of the probe. In one example, the oligonucleotide is attached to the solid support by the 5 'end. However, one skilled in the art can determine whether the use of the 3 'or 5' end of the oligonucleotide is suitable for attachment to a solid support. In general, the internal complementarity of oligonucleotide probes within the 3 'and 5' end regions determines binding to the support. Alternatively, the oligonucleotide probe can be attached to the support by a sequence other than the sequence of BF, C2, and / or CFH, such as an oligonucleotide or other molecule that acts as a spacer or linker to the solid support.

  In another example, the array includes protein sequences that are at least one BF protein, C2 protein, and / or CFH protein (or a gene, cDNA, or other that includes one of the described sequences) Polynucleotide molecules, or fragments thereof), fragments of such proteins, or antibodies specific for such proteins or protein fragments. The proteins or antibodies forming the array can be linked directly to the support. Alternatively, the protein or antibody can be attached to the support by a spacer or linker to the solid support.

  Abnormalities in BF protein, C2 protein, and / or CFH protein are detected using a binding agent that is optionally detectably labeled, eg, specific for BF protein, C2 protein, and / or CFH protein. be able to. Thus, in certain instances, detection of an abnormality involves contacting a sample from a subject with a binding agent specific for BF protein, C2 protein, and / or CFH protein, and binding of the sample to the binding agent. Measuring the amount of BF, C2 and / or CFH protein present in the sample, but in this case predisposed to developing AMD The amount of BF protein, C2 protein, and / or CFH protein present in a similar sample from a non-subject, or BF protein, C2 protein, and / or in a similar sample from a subject not predisposed to develop AMD Or BF protein, C2 tamper in the sample compared to the standard amount of CFH protein. Quality, and / or have different amounts of CFH protein, BF molecule is an abnormal of C2 molecule, and / or CFH molecule.

  In certain instances, the microarray material is made from glass (silicon dioxide). Suitable types of silicon dioxide for solid supports include, but are not limited to, aluminum silicate, borosilicate, silica, soda lime, zinc titania, and fused silica (eg, Schena, Microarray Analysis. John Wiley). & Sons, Inc, Hoboken, New Jersey, 2003). Nucleic acid can be attached to the surface of glass by a method well known in the art, for example, by a surface treatment using an organic polymer as a raw material. Specific examples are polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluoroethylene, polyvinylidene difluoride, polyfluoroethylene-propylene, polyethylene vinyl alcohol, polymethylpentene, poly Chlorotrifluoroethylene, polysulfurone, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, ethylene acrylic acid, thylene methacrylate, and mixtures of these (US Pat. No. 5, No. 985,567, which is incorporated herein by reference), organosilane compounds that provide chemically active amine groups or aldehyde groups, microarray epoxies or polymers. Including lysine treated, without limitation. Another example of a solid support surface is polypropylene.

  In general, suitable properties for materials that can be used to form a solid support surface include: That is, since it is easily surface activated, when activated, the surface of the support can covalently bind biomolecules such as oligonucleotides; suitable for “in situ” synthesis of biomolecules; In the region on the support that is not occupied by the oligonucleotide, it is less likely to cause non-specific binding, or when non-specific binding occurs, such as without removing the oligonucleotide. For example, a simple substance can be easily removed from the surface.

  In one example, the surface treatment agent is an amine-containing silane derivative. Nucleic acid attachment to the amine surface occurs through the interaction of negatively charged phosphate groups on the DNA backbone and positively charged amino groups (Schena, Microarray Analysis. John Wiley & Sons, Inc, Hoboken). , New Jersey, 2003, which is incorporated herein by reference). In another example, a reactive aldehyde group is used as a surface treatment agent. Attachment to the aldehyde surface is performed by adding a 5'-amine group or an amino linker to the target DNA. Bonding occurs when the unbonded electron pair on the amine linker acts as a nucleophile that attacks the positive carbon atom of the aldehyde group.

  A variety of array formats can be used in accordance with the present disclosure. An example includes a linear array of oligonucleotide bands, commonly referred to in the art as dipsticks. Another suitable format includes a two-dimensional pattern (such as 4096 squares in a 64 × 64 array) in separate cells. As those skilled in the art will appreciate, other array formats are equally suitable for use, including but not limited to slotted (rectangular) and circular arrays (see US Pat. No. 5,981,185, which is hereby incorporated by reference). Which is incorporated herein by reference). In one example, the array is formed on a polymer medium in the form of a thread, a film, or a film. An example of an organic polymer medium is a polypropylene sheet with a thickness of about 1 mm (0.001 inch) to about 20 mm, although the thickness of the film is not critical and can vary over a fairly wide range. Biaxially oriented polypropylene (BOPP) film is now specifically disclosed for preparing arrays. In addition to its durability, BOPP film exhibits low background fluorescence. In a particular example, the array is a nucleic acid array using solid phase allele-specific oligonucleotides (ASOs).

  The array formats of the present disclosure can be included in a variety of different types of formats. “Format” includes any format capable of fixing a solid support, such as microtiter plates, test tubes, inorganic sheets, dipsticks, and the like. For example, when the solid support is a polypropylene yarn, one or more polypropylene yarns can be attached to a plastic dipstick type device, and the polypropylene membrane can be attached to a slide glass. The specific format is not important per se. What is needed is that the solid support can be affixed without affecting the functional behavior of the solid support, or any biopolymer adsorbed thereto, and the format (dipstick Or a glass slide) is stable to any material (such as clinical samples and hybridization solutions) into which the device is introduced.

  The arrays of the present disclosure can be prepared by a variety of methods. In one example, oligonucleotide or protein sequences are synthesized separately and then attached to a solid support (see US Pat. No. 6,013,789, which is incorporated herein by reference). In another example, the sequences are synthesized directly on a solid support to provide the desired array (see US Pat. No. 5,554,501, which is incorporated herein by reference). Methods suitable for covalently attaching oligonucleotides and proteins to a solid support, and methods suitable for directly synthesizing oligonucleotides or proteins on a support are well known to those skilled in the art, and a summary of suitable methods. Matson et al. (1994) Anal. Biochem. 217: 306-10. In one example, oligonucleotides are synthesized on a support using conventional chemical techniques for preparing oligonucleotides on a solid support (eg, PCT Publications 85/01051 and 89/10977, or the United States). No. 5,554,501, etc., each of which is incorporated herein by reference).

  Appropriate arrays can be made by anchoring the four base precursors in a predetermined pattern using an automated means of synthesizing oligonucleotides within the cells of the array. In essence, a multi-channel automated chemical delivery system is used to create oligonucleotide probe populations in parallel rows (as many as the number of channels in the delivery system) across the substrate. After the synthesis of the oligonucleotide in the first direction is complete, the substrate can be rotated 90 degrees to allow the synthesis to proceed within the second set of columns perpendicular to the first set. This process creates a multichannel array that produces a plurality of separate cells at the intersection.

  In certain instances, the oligonucleotide probes on the array include one or more labels that allow detection of the hybridization complex of oligonucleotide probe: target sequence.

Kits The present disclosure provides kits that can be used to determine whether a subject, eg, an otherwise healthy human subject, has a genetic predisposition to AMD. Such a kit allows to determine whether a subject has one or more gene mutations or polymorphisms within the BF, C2 or CFH gene sequence.

  These kits are useful for determining whether or not at least one polymorphism is present in the BF gene, C2 gene, or CFH gene of interest, eg, BF, C2 identified herein. Alternatively, a probe or primer that selectively hybridizes to the polymorphic sequence of CFH is included. Such kits can be used with the methods described herein to determine a subject's BF, C2, or CFH genotype or haplotype.

  Providing oligonucleotide probes and / or primers in the form of kits used to detect specific BF, C2, or CFH sequences in a subject, eg, SNPs or haplotypes described herein You can also. In such a kit, an appropriate amount of one or more oligonucleotide primers is provided in one or more containers. These oligonucleotide primers can be suspended, for example, in an aqueous solution or provided as freeze-dried or lyophilized powder. The container supplying the oligonucleotide may be any conventional container capable of retaining the shape as supplied, for example, a microcentrifuge tube, an ampoule, or a bottle. Depending on the intended use, the primer pairs can be pre-measured in separate tubes, which are typically disposable, or equivalent containers. With such a preparation, a sample to be examined for the presence of a BF, C2, or CFH polymorphism can be added to each tube for direct amplification.

  The amount of each oligonucleotide primer supplied in this kit may be any appropriate amount, and is determined, for example, according to the market to which the product is aimed. For example, if the kit is tailored for research or clinical use, the amount of each nucleotide primer provided will be sufficient to initiate several PCR amplification reactions. Those skilled in the art know the amount of oligonucleotide primer suitable for use in a single amplification reaction. General guidelines include, for example, Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (In Molecular Claring A: L New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992).

  To facilitate in vitro amplification of sequences encoding BF, C2 or CFH, such as the specific target BF, C2 or CFH genes or their 5 ′ or 3 ′ neighboring regions In addition, the kit may contain three or more kinds of primers.

  In some embodiments, the kit performs a nucleotide amplification reaction, such as, for example, a DNA sample preparation reagent, a suitable buffer (eg, a polymerase buffer), salts (eg, magnesium chloride), and deoxyribonucleotide (dNTP). It may also contain the reagents necessary to carry out.

  In addition, the kit can include labeled or unlabeled oligonucleotide probes for use in detecting BF, C2, or CFH polymorphisms or haplotypes. In certain embodiments, these probes will be specific for potential polymorphic sites that may be present in the amplified target sequence. Suitable sequences for such probes include one or more of the identified polymorphic sites such that the probe sequence is complementary to the polymorphic site and the surrounding BF, C2, or CFH sequence. Any sequence containing. As an example, such a probe is at least 6 nucleotides in length and a polymorphic site is present at any position in the length of the probe. In order to ensure specificity, it is often beneficial to use longer probes. Thus, in some embodiments, the probe is at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, or more.

  In this kit, it may also be beneficial to provide one or more control sequences for use in the amplification reaction. The design of appropriate positive control sequences is well known to those skilled in the appropriate art. By way of example, a control sequence may comprise a human (or non-human) BF, C2, or CFH nucleic acid molecule having a known sequence at one or more target SNP sites as described herein. Good. Controls can also include non-BF, non-C2, or non-CFH nucleic acid molecules.

  In some embodiments, the kit comprises some or all of the reagents necessary to perform the RT-PCR method in an in vitro amplification reaction, such as an RNA sample preparation reagent (eg, an RNase inhibitor). A suitable buffer (eg, polymerase buffer), salts (eg, magnesium chloride), deoxyribonucleotide (dNTP), and the like.

  In addition, such kits can include labeled or unlabeled oligonucleotide probes for use in the detection of target sequences amplified in vitro. A suitable sequence for such a probe is any sequence between the annealing sites of the two oligonucleotide primers provided, such that the sequence to which the probe is complementary is amplified during the PCR reaction. Will. In certain embodiments, these probes are specific for potential polymorphisms that may be present in the amplified target sequence.

  It may also be beneficial to provide one or more control sequences for use in the RT-PCR reaction in the kit. The design of appropriate positive control sequences is well known to those skilled in the appropriate art.

  Also included are kits for detecting or analyzing BF, C2, or CFH protein expression (eg, overexpression or underexpression, or expression of a particular isoform, etc.). Such a kit comprises at least one target protein-specific binding agent (eg, a polyclonal or monoclonal antibody or antibody fragment that specifically recognizes BF protein, C2 protein, or CFH protein, or BF protein, C2 protein, or A specific polymorphic form of CFH protein) and a sample comprising at least one control (a predetermined amount of target BF, C2, or CFH protein, or a predetermined amount of BF, C2, or CFH protein) Etc.). The agent that specifically binds to the BF, C2, or CFH protein and the control can also be placed in separate containers. Such antibodies may have the ability to distinguish polymorphic forms of BF protein, CD protein, and / or CFH protein.

  A kit that detects expression of a BF protein, C2 protein, or CFH protein, or isoform can also include a BF, C2, or CFH: binding agent complex, eg, the binding agent can be detected. May be labeled. If the detectable binding agent is not labeled, it can be detected, for example, by a secondary antibody or protein A, which is provided in one or more separate containers, depending on the kit. You can also. Such techniques are well known.

  Additional components of a specific kit may include instructions for performing the assay. The instructions allow the tester to measure the expression level of BF, C2, or CFH. The kit may also contain reaction vessels and auxiliary reagents such as chromogens, buffers and enzymes. The instructions may provide a calibration curve or correction diagram for comparison with a determined value (eg, an experimentally measured value).

  There is also a kit that makes it possible to distinguish between homozygous and heterozygous individuals for specific SNPs (or haplotypes) of the BF gene, C2 gene or CFH gene as described herein Provided. Examples of such kits include Nickerson et al. (1990) Proc. Natl. Acad. Sci. U. S. A. 87: 8923-8927 provides the materials necessary to perform an oligonucleotide ligation assay (OLA). In a specific embodiment, these kits are one or more designed to detect polymorphisms within a BF, C2, or CFH sequence of interest, as described herein. Includes microtiter plate assay. The instructions included in these kits allow the tester to determine whether a particular BF, C2, or CFH allele is present and whether it is homozygous or heterozygous. Will be able to. It may also be beneficial to provide one or more control sequences for use in the OLA reaction with this kit. The design of appropriate positive control sequences is well known to those skilled in the appropriate art.

  The kit may include the use of several assay formats, including nucleic acid binding, such as binding to filters, beads, microtiter plates, and the like. Techniques include dot blotting, RNA blotting, DNA blotting, PCR, RFLP and the like.

  A microarray-based kit is also provided. These microarray kits may be useful in genotyping analysis. Generally, these kits include one or more oligonucleotides provided on a substrate, eg, immobilized at an addressable site. The kit also includes normally written instructions that help the user search the array. Such instructions can optionally be provided on computer readable media.

  The kit can further include one or more buffers for use in analyzing the provided array. For example, such a buffer may be a low stringency wash, a high stringency wash, and / or a stripping solution. These buffers can be provided together. That is, each buffer solution container is large and may hold a buffer solution sufficient to perform several times of probing processing, washing processing, or peeling processing. Alternatively, the buffer solution can be provided as an equal volume solution that is pre-weighed according to the size and shape of the array included in the kit. Certain kits may provide one or more containers in which array-probing reactions can be performed.

  The kit further includes one or more containers of detection molecules, such as antibodies or probes (or a mixture of antibodies, a mixture of probes, or a mixture of antibodies and probes) to detect biomolecules captured on the array. You may go out. The kit can also include labeled probe probes, labeled or unlabeled, for internal testing of either labeling or array probing, or both. Control probe molecules can be provided, for example, as a suspension in an aqueous solution or as a freeze-dried or dry-frozen powder. The container containing the control may be any conventional container capable of holding the supplied form, such as a microcentrifuge tube, ampoule, or bottle. Depending on the intended use, the control probe can be provided in a pre-weighed single-use amount in a separate, generally disposable tube or equivalent container.

  The amount of each control probe supplied in the kit can be any specific amount, for example, depending on the market to which the product is aimed. For example, if the kit is tailored for research or clinical use, sufficient control probes will be provided to analyze the array several times for control. Similarly, where multiple control probes are provided in one kit, the specific probes provided are provided to match the market and accompanying kit. In certain embodiments, a plurality of different control probes are provided in a single kit, but each control probe is derived from a different type of analyte present on the associated array (eg, A kit that provides both eukaryotic and prokaryotic specimens can provide a prokaryotic-specific control probe and another eukaryotic-specific control probe).

  In some embodiments of the invention, it may also include reagents necessary to perform one or more probe labeling reactions. Specific reagents included depend on the type of probe molecule (eg, DNA or RNA) and the labeling method (eg, radiolabel incorporated in the probe synthesis process, bindable fluorescent tag, etc.) Should be chosen to satisfy.

  Additional kits are provided for labeling the probe molecules used to assay the arrays provided herein. Such a kit can optionally include an array to be assayed with probe molecules so labeled.

  The present disclosure is specifically illustrated by the following non-limiting examples.

Example 1
Materials and Methods Subjects: In this study, two independent groups of AMD cases over 60 years old, European and American offspring, and age-matched controls were used. These groups consisted of 350 unrelated clinically identified AMD (average age 79.5 +/− 7.8 years) reported by the University of Iowa and 114 unrelated controls Individuals (mean age 78.4 +/− 7.4 years; age and ethnicity matched), and 548 unrelated clinically confirmed AMD reported from Columbia University (mean age 71. 32 +/− 8.9 years) and 275 unrelated, age-ethnic matched controls (mean age 68.84 +/− 8.6 years). Subject was examined by a skilled ophthalmologist.

  Stereoscopic fundus photographs are described in Hageman, 2005, supra; Bird et al. (1995) Surv. Ophthalmol. 39: 367-74; and Klaver et al. (2001) Invest. Ophthalmol. Vis. Sci. 42: 2237-41, graded according to a standardized classification system. The controls showed no signs of macular disease and none were known to have a family history of AMD (Stage 0 and 1a). When recruiting AMD subjects, they were subdivided into phenotypic categories based on their severe eye classification. Genomic DNA was prepared from peripheral blood leukocytes using the QIAamp DNA Blood Maxi kit (Qiagen, Valencia, CA).

  Experiments were performed according to protocols approved by the Institutional Review Board at Columbia University and the University of Iowa. Informed consent was obtained in advance from all subjects.

  Immunohistochemistry: Anderson et al. (2002) Am. J. et al. Ophthalmol. 134: 411-31, the posterior poles were processed into sections and labeled with an antibody against Ba factor (Quidel). Adjacent sections were incubated with secondary antibody alone and used as a control. As previously described (Anderson et al., 2002, supra), several immunolabeled specimens were prepared and observed with a confocal laser scanning microscope.

  Mutation screening and analysis: Mutants were examined for intron regions adjacent to the coding regions of BF and C2 using SSCP analysis, denaturing high performance liquid chromatography (DHPLC), and direct sequencing. Primers for analysis by SSCP, DHPLC, and DNA sequencing were designed using Mac Vector software (San Diego, CA) to amplify each exon and the adjacent intron region. Allikmets et al. (1997) Science 277: 1805-1807, and Hayashi et al. (2004) Ophthalmic Genet. 25: 111-9, amplicons generated by PCR were screened for sequence variation. All mutations detected by SSCP and DHPLC were confirmed by bi-directional sequencing according to standard protocols.

  Genotyping: Single nucleotide polymorphisms (SNPs) were discovered by data mining (Ensembl database, dbSNP; Celera Discovery System) and sequencing. Variant assays more frequently than 10% within the assay population were purchased from Applied Biosystems as Validated, Inventored SNP Assays-on-Demand, or submitted to Applied Biosystems Assays-By-Design Pipeline. The technology utilized was the same as that described in Hageman et al., 2005, supra. Briefly, 5 ng of DNA was amplified for 50 cycles on an ABl 9700 384-well thermal cycler and the plate was read on an Applied Biosystems 7900 HT Sequence Detection System.

Statistical analysis: To perform a contingency table analysis as described in Hageman et al., 2005, supra, and Klaver et al., 2001, supra, genotypes are listed in Microsoft Excel and listed in SPSS (SPSS, Inc.). submitted. SAS / Genetics (SAS Institute, Inc., Cary, NC) was used to confirm the fit to Hardy 7-Weinberg equilibrium, and all SNPs were found to be p <0. Cut through the cutoff value of 05. In order to estimate the haplotype, we have the Cambridge Medical Institute website http: // www-gene. cimr. cam. ac. Sncap downloaded from uk / clayton / software / (from David Clayton; obtained from Cambridge Institute for Medical Research, Cambridge, United Kingdom), SNPEM (from Dr. Nichol, Dr. Nichol, and D. Nichols). PHASE version 2.11 (available by Matthe Stephens; available from University of Washington, Seattle, WA and his website www.stat.washington.edu/stephens/software.html). The haplotype analysis method used is primarily to obtain an estimate of the haplotype using an expectation maximization (EM) algorithm or a Gibbs sampling algorithm, and secondly, within the region of linkage disequilibrium. The third is to identify the htSNPs that represent the smallest set of information, and third, to evaluate that they are significantly associated with AMD. Linkage disequilibrium was assessed using an image tool available on the Innate Immunity PGA website (www.inimmunity.net) (not shown). All p values are two-sided tested and X2 values are displayed as asymptotic significance. The total type I error rate (α) is conducted at Benjamini and Hochberg (1995), conducted at the Innate Immunity PGA website (https://internalimmunity.net/IIPGA2/Bioinformatics/multipletestfdrform). R. Stat. Soc. Ser. B57: calculated retrospectively using the method of 289-300, but less than 2 × 10 ⁻³ .

  The haplotypes with significance were subjected to a sort test by SNPEM and PHASE. The protected SNP model shown in FIG. 2A is presented in Exemplar 2.2 (available on the Sappio Sciences website at http://www.sapiosciences.com), and this software allows the three SNP models shown in FIG. A statistical assessment was made of the goodness of fit to the databases (Iowa, Colombia, and mixed). The creation of a model (shown in FIG. 2C) derived from the genetic algorithm (GA) included the Exemplar software. The GA option values were set as follows. That is, a 1500 AND / OR model with a model size of 5 or less (16 genotypes are possible) was repeated 15 times. Further details of the implementation of the genetic algorithm and the significance test are contained in Example 2.

  For Columbia, Iowa, and mixed data recoded with (+) and without (-) minor alleles using the SPSS version 14.0 statistical package with appropriate modules Classification and regression tree analysis were performed. The model was automatically generated using each of the three data sets that incorporated both the CFH and C2 / BF loci as contributing factors to independent results.

Results In approximately 90 AMD cases and 90 controls obtained from a cohort identified at Columbia University, a total of 18 BF exons were first included by denaturing HPLC, including the adjacent intron region of 50-80 bp. And analyzed. When 17 types of sequence variants including 8 types of missense mutations were identified, the L9H (rs4151667) allele and the R32Q (rs641153) allele were more frequent in the subject than in the case (Table 1). A haplotype-tagging SNP (htSNP) within BF and its adjacent homolog C2 has been identified (FIG. 1) and genotyped in a Columbia University cohort of 548 AMD cases and 275 controls It was. These analyzes revealed four variants that were significantly associated with AMD. The L9H mutant in BF was in almost complete linkage disequilibrium (LD) with the E318D mutant in C2 (rs93332739) but was very protective against AMD (X2 = 13.8 P = 0) 0.0002, OR = 0.37 [95% CI = 0.18-0.60]). The R32Q allele in BF is in nearly complete LD with the rs547154 SNP in intron 10 of C2, which was also very protective (X2 = 33.7, P = 6.43 × 10-9, OR = 0.32 [95% CI = 0.21 to 0.48]).

  These findings were confirmed by genotyping of an independent cohort of 350 cases and 114 controls obtained at the University of Iowa. For example, the C2 E318D / BF L9H SNP pair was significantly associated with AMD in this cohort (X2 = 10.6, P = 0.0012, OR = 0.34 [95% CI = 0.18− 0.67]). To analyze haplotypes across the C2 and BF loci, the data from the two cohorts were combined (Table 2). The common haplotype (H1, FIG. 1) posed a significant risk for AMD (X2 = 10.3, P = 0.0013, OR = 1.32 [95% CI = 1.1-1.6). ]). The haplotype (H7) tagged with the BF R32Q SNP is very protective against AMD compared to all other haplotypes (X2 = 26.9, P = 2.1 × 10 −7). , OR = 0.45 [95% CI = 0.33 to 0.61], and a haplotype (H10) containing C9 E318D / BF L9H was also significantly protective (X2 = 21.6, P = 3.4 × 10 −6, OR = 0.36 [95% CI = 0.23 to 0.56]) (FIG. 1) When the H1 haplotype is used as a reference haplotype, H7 (X2 = 29. 6, OR = 0.42 [0.32-0.58]) and H10 (X2 = 24.9, OR = 0.33 [0.21-0.52]) slightly more significant The result was a solution by the SNPEM program. Analysis also revealed that the same haplotype was significantly associated with the disease, supporting the hypothesis that alleles in the C2 gene and / or BF gene could predict risk for AMD. Individuals who were homozygous for H7, H10 or 7/10 complex heterozygotes were found in 3.4% of controls but only 0.77% in cases (X2 = 12.2, P = 0.00048, OR = 0.22 [0.087-0.56]) The odds ratio for a subject with two protective alleles is one Nearly half the odds ratio of subjects with protective alleles, consistent with the codominant model.

  Observed associations were observed when the entire AMD subject cohort was compared to controls or separated by major AMD subtypes such as early AMD (eAMD), choroidal neovascularization (CNV), and geographic atrophy (GA). The analysis was very significant. The GA group (a total of 133 subjects from the two cohorts), as the case may be, is similar to our observations related to CFH (Hageman et al., 2005, supra). Deviated. Specifically, haplotypes tagged with the R32Q allele showed the strongest protection against this disease. That is, when the GA group was compared with the control, the OR was 0.22, whereas the remaining AMD sample was 0.45 when subjected to the same analysis. This shift may be significant in that there are various etiologies for this disease, but was not statistically significant, probably due to the small number of GA cases (trust Overlapping sections).

  The composite analysis was performed by first stratifying the subject according to the state of the Y402H allele of CFH. The protection conferred by C2 / BF is strongest with the CFH 402H homozygote (OR = 0.27), moderate with the 402H / Y heterozygote (OR = 0.36), and the 402Y homozygote. It was the weakest (OR = 0.44). However, confidence intervals overlap for all of these estimates. This result was mainly due to the tendency of the highest frequency of C2 / BF protective alleles in the 402H homozygote (“risk” genotype). Forty percent of these subjects in the control cohort had at least one protective allele. In contrast, in controls that were 402H / Y or 402Y, the frequency of C2 / BF protection gradually decreased (32% and 26%, respectively). In other words, individuals who are at high risk due to the CFH genotype do not develop AMD and frequently have a protective allele at the C2 / BF locus.

In order to identify possible combinations of CFH and C2 / BF SNPs that are protective against AMD, as suggested by individual SNP analysis, analysis of available data can be performed in two ways: This was done by an empirical handmade model and then by a machine-learned model using Exemplar software (FIG. 2). The first model was a hypothetical (handmade) model that was created by examining the data empirically (Figure 2A). The model description is shown as Panel A and is understood to present four possible combinations of genotype combinations that protect against AMD (the combination that results in a model that is “true”) . When this model was applied to the sample, the distribution shown in Panel B for each cohort and mixed cohort was obtained (FIG. 2B). The percentage in cases is the proportion of cases that the model is false, in other words, those who did not receive protection as represented by the model. The percentage in the control is the percentage of the control who has the protective factor represented by the model and means that the model is true. These distributions are subjected to a significance test by Fisher's exact test, with p-values of P = 0.00237, P = 4.28 × 10−8, and P = 7.90 × 10−10, respectively. Prove that there is. After this, the Exemplar software was run to create a “best fit” defense model for the data using a machine learning method called a genetic algorithm. That is, we tested the hypothesis that machine learning software can outperform handmade models. The model was trained for the Colombian cohort and the resulting best fit model was retained and then applied to the Iowa cohort as a validation test (out-of-sample verification) for an independent cohort. These models were applied to a mixed sample set, and the best model obtained is shown in Figure 2C, which shows four distinct (or combined) genotypes (ie, " The results of this model for each of the Iowa, Columbia, and mixed cohorts are shown in Figure 2D. Significance by Fisher's exact test for these distributions A sex test was performed, and each of the p values was P = 7.49 × 10 ^{− 5} , P = 2.97 × 10 ⁻²² , and P = 1.69 × 10 ⁻²³ Randomized case and control designations and 3000 dataset replacements This method was further validated by: the actual data was more significant than any of these substitutions.

  In summary, when these haplotypes with mutations in CFH were combined and analyzed by the Exemplar software, 56% of healthy controls had at least one protective CFH haplotype or C2 / BF haplotype, but 74% It was found that AMD subjects do not have any protective haplotypes at these loci. Examining this data, about 60% of the risk type in the case and 65% of the protective type in the control are due to the action of the CFH locus, the rest (40% and 35%, respectively) are C2 / BF. It can be seen that this is due to the locus. The machine learning model is superior to the handmade model and can predict clinical results significantly more correctly. Classification and regression tree (C & RT) analysis created a tree similar to the genetic algorithm analysis with results supporting the role of C2 / BF in AMD. Using the Colombian dataset alone, the C & RT model accounts for 37% of cases due to the presence of the C2 / BF allele, but with Iowa it accounts for 36% and the mixed analysis has a slightly lower effect of 27%. Indicated. All of these estimates are consistent with the 35/40% C2 / BF locus contribution estimated from genetic algorithm analysis. Details and specific analysis of these methods are described in Example 2.

  BF and C2 are expressed in the neural retina, RPE, and choroid. From human donor eyes, isolated from eyes with AMD (2 donors, 67 and 94 years old) and eyes without AMD (2 donors, 69 and 82 years old) Appropriately sized PCR amplicons for BF and C2 gene products were detected from the engineered RPE, RPE / choroid complex, and neural retina (data not shown). BF protein was present in the drusen of the eye inside the Bruch's membrane, and less prominently in the choroid parenchyma (FIG. 3A). The immunoreactivity of Ba (a peptide derived from BF) was less pronounced, but was clearly present in the spots associated with RPE cells and throughout Bruch's membrane (FIG. 3B). The distribution of BF is similar to that of C3 (FIG. 3C), both of which are essentially identical to those of CFH and C5b-9 (Hageman et al., 2005, supra).

  In summary, these data indicate that variants of the complement pathway related genes C2 and BF are significantly associated with AMD. A protective haplotype at the C2 / BF locus contains a non-synonymous SNP in the BF gene, which is an important activator of the second complement activation pathway. Available data supported the hypothesis that the AMD phenotype could be regulated by abnormal BF activity. Indeed, it is clear that BF protein containing glutamine at position 32 (obtained from one of the two BF SNPs that tag the protective haplotype) has less hemolytic activity than the more frequent arginine type 32. (Lokki and Koskimies (1991) immunogenetics 34: 242-6). The same study has not demonstrated a functional effect on the R32W mutant, but in this study there was no association with AMD. Based on these data, we suggest that activators with reduced enzyme activity result in a reduced risk for chronic complement reactions that can lead to drusen formation and AMD. . This hypothesis is consistent with our presentation that insufficient inhibition of the second complement cascade by mutations in CFH results in chronic damage at the retinal pigment epithelium / Bruch membrane interface (Hageman et al., 2005, supra; Anderson, 2002, supra; Hageman, 2001, supra). Another BF htSNP, L9H, is located in the signal peptide. Although the functional consequences of this variant are not directly apparent, this variant may regulate BF secretion.

  Data on genes and functions suggest that mutations in BF are likely responsible for the observed association with AMD. This demonstrates that the mutants that tag two haplotypes in BF are non-conservative mutants, one of which has a direct functional relevance (reduced hemolysis). In contrast, the mutant in C2 is based on the fact that it is a conservative mutation and is an intron SNP. In addition, BF is directly involved in the second pathway, which is a pathway involving CFH. However, in particular, since C2 and BF both control C3 production, the direct role of C2 cannot be ignored. C2 and BF have almost identical molecular structures, such as having a serine protease domain at its carboxy terminus and three CCP modules at its amino terminus. Further evidence that BF is a gene involved in the pathogenesis of AMD comes from studies of drusen composition. The majority of proteins (CFH, BF, etc.) involved in the second pathway are present in drusen, whereas their analogs are not found in classical pathways such as C2 and C4 (Mullins et al. (2000) Faseb J 14: 835-46; Crabb et al. (2002) Proc. Natl. Acad. Sci. USA 99: 14682-7). These data further suggest that SNPs in the C2 gene are associated with AMD due to extensive LD with BF.

  Several common functional variants in both C2 and BF have been described (Davis and Forristal (1980) J. Lab. Clin. Med. 96: 633-9; Raum et al. (1979) Am. J. Hum. Genet. 31: 35-41; Alper et al. (2003) J. Clin. Immunol. 23: 297-305), most of which are rare. Judging from the resequencing data for both genes available on the Seattle SNP project website (www.pga.mbt.washington.edu/), all missense alleles with a frequency higher than 2% in the European population Was analyzed. Furthermore, after full-length sequencing of several HLA haplotypes, including examples of our haplotypes H2, H5, and H7, no further non-synonymous variants have been found in either gene ( Stewart et al. (2004) Genome Res. 14: 1176-87).

  Because C2 and BF are present at the HLA locus along with many other genes related to inflammation, it is necessary to consider the association observed in this study may be due to LD with adjacent genes There is. (Larsen and Alper (2004) Curr. Opin. Immunol. 16: 660-7). However, five types of evidence suggest that the C2 / BF locus is a major contributor to the observed association. First, in the HapMap data, only moderate LD can be seen between C2 / BF and the adjacent class III locus. Next, it is mentioned that the MHC class II locus and the BF haplotypes H7 and H10 do not show strong LD. Third, in a whole genome scan performed by Klein et al. (2005) Science 308: 385-9, the MHC locus did not show a statistically significant association with AMD. Their analysis using the Affymetrix Mapping 100K Array included 80 SNPs across the MHC locus, but did not include any of the 8 SNPs typed in this study (https: // /Www.affymetrix.com/analysis/netaffx/index.affx). Fourth, recombination rates estimated from HapMap data indicate that there is a high recombination region on both sides of the C2 / BF locus (Myers et al. (2005) Science 310: 321-324). ). Finally, one published study on MHC in AMD showed a moderate for class I locus B * 4001 (P = 0.027) and class II locus DRB1 * 1301 (P = 0.0099). Protection has been shown (Goverdhan et al. (2005) Invest. Ophthalmol. Vis. Sci. 46: 1726-34). Since the protective alleles identified in this study are associated with AMD with substantially higher statistical significance, the C2 / BF association may be associated with these loci and / or other loci of MHC. The possibility of being due to LD is very low.

  Table 1. Sequence variants in the BF gene detected by DHPLC screening

表２．混合コロンビア・アイオワコホートにおけるＣ２／ＢＦ変異体の関連性解析

Table 2. Association analysis of C2 / BF mutants in a mixed Columbia Iowa cohort

実施例２．Ｅｘｅｍｐｌａｒ統計学法
Ｓａｐｉｏ Ｓｃｉｅｎｃｅは、ＮＣＩと共同して遺伝子型判定データを解析した。ＮＣＩが、全部で約１３６０の試料を、遺伝子型判定された１０の２対立遺伝子ＳＮＰとともに提供した。このデータは、対立遺伝子を数値表現したものとともにＳａｐｉｏに提示された。このデータを、対立遺伝子を遺伝子型の数値表現に変えることによって（ＡＡについては「１１」が「１」となり、ＡＢについては「１２」が「２」となり、ＢＢについては「２２」が「３」となり、「００」はノーコールであり、「０」に変換された）、Ｅｘｅｍｐｌａｒを行いやすいフォーマットに変換し、また試料の重複を排除するためにスクリプトを書いた。表現型は加齢性黄斑変性症（ＡＭＤ）であった。同定されたＡＭＤにはいくつかのサブクラスがあったが、本解析では、データを全体として用いて、さまざまなＡＭＤ表現型の基となる共通した遺伝子型があるか否かを判定した。

Example 2 Exemplar Statistics Method Sapio Science analyzed genotyping data in collaboration with NCI. NCI provided a total of approximately 1360 samples with 10 biallelic SNPs that were genotyped. This data was presented to Sapio along with a numerical representation of the allele. By changing this allele to a numerical representation of the genotype (“11” is “1” for AA, “12” is “2” for AB, and “22” is “3” for BB. “00” is a no call and converted to “0”), and the script was written to convert the exemplar into an easy-to-use format and eliminate sample duplication. The phenotype was age related macular degeneration (AMD). There were several subclasses of identified AMD, but in this analysis, the data as a whole was used to determine whether there were common genotypes that underlie various AMD phenotypes.

Sapio Science analyzed the supplied data using the Exemplar Genotyping Analysis Suite. Exemplar performs several association-based analyzes for case-control studies. The modules used for this analysis are as follows.
Genetic Algorithm Module (GA Module)-This module performs a machine learning method to find logical combinations of experiments based on SNPs (models).
Relevance Experiment Module (AS Module)-This module has many such as Chi-square, Yates method, Fisher's exact test, odds ratio, relative risk factor, linkage disequilibrium, D ', r2, and haplotype estimation Calculate useful statistics.

  Exemplar generally finds models that correlate with phenotypes. In other words, the model predicts not the defense from the phenotype, but the factors that contribute to obtaining the phenotype, but it is possible to infer the defense factor from this model. For example, if a model indicates that a sample with RS001 is BB or RS001 is AB correlates with its phenotype, a sample with RS001 is AA will be protected from that phenotype I can guess it will be done.

  The Explorer model is a logical combination of SNPs. This model can be hand crafted to test hypotheses or an attempt can be made to find a highly useful model using genetic algorithms. Genetic algorithms are machine learning methods that are good at finding patterns in large data regions. GA uses the 2/3/3 validation method. This is done by randomly assigning 2/3 of the cases and controls to the training set. And GA learns the model regarding this training set. At the end of the learning period, the most efficient model is applied to the test set (the remaining 1/3 of the data). Through testing and training, the most efficient model is returned to the user. In this study, even if only a small number of SNPs were examined, because of the large number of samples, it was difficult for humans to effectively distinguish patterns that could be applied through all data. For this reason, GA was used to find more complex patterns that were more useful. The benefit of these types of models over traditional approaches is that many loci can be taken from the entire genome when making predictions. This allows one model to identify what constitutes a complex interaction of polymorphisms that often correlate with results.

  This study was unique in that it focused on finding a protective model against AMD. This deviates from the usual Exemplar method of finding an additive model, so changes had to be made to the input data. Just telling Exemplar that the case is the control and vice versa, it learns a model that reveals why a sample did not acquire that phenotype. That is, an attempt was made to find a combination of SNPs that provided protection.

  Study Group Information: Data were provided from two separate cohorts: the Iowa and Columbia cohorts. The Colombian data were a large group with a total of about 830 samples, with 560 cases and 270 controls. The Iowa Cohort had a total of about 529 samples with 414 case subjects and 115 controls. Because there were two sample groups, it was possible to build a model using the GA module in one cohort and test the out-of-sample efficiency of the resulting model in the other cohort. there were.

  Study results: A number of statistics were generated for each SNP / genotype in the input data set. Statistics were generated by creating a 2x2 contingency table and counting genotypes properly (this is not an allele count, but collapses two uncalculated genotypes into one value) Note that this is a genotype count). The values for each cell in the 2 × 2 contingency table are listed in the table under the headings Case (true), Case (false), Control (true), Control (false). All statistics were two-sided calculations.

  Tables 3-6 show in parallel the statistics for the Iowa and Columbia cohorts. Note: In the “Category” column, 1 corresponds to AA, 2 corresponds to AB, and 3 corresponds to genotype. Table 7 shows the statistics for the mixed cohort.

  Table 3. Chi-square statistics for Colombia and Iowa

表４．コロンビアとアイオワを並列させたカイ二乗イェーツ統計値

Table 4. Chi-square Yates statistics with Colombia and Iowa in parallel

表５．コロンビアとアイオワを並列させたフィッシャー正確確率統計値

Table 5. Fisher exact probability statistics for Colombia and Iowa

表６．コロンビアとアイオワを並列させたオッズ比統計値

Table 6. Odds ratio statistics for Colombia and Iowa in parallel

表７．混合コホートのカイ二乗統計値

Table 7. Chi-square statistics for mixed cohorts

表８．混合コホートのカイ二乗イェーツ統計値

Table 8. Chi-square Yates statistics for mixed cohorts

表９．混合コホートのフィッシャー正確確率統計値

Table 9. Fisher exact probability statistics for mixed cohorts

表１０．混合コホートのオッズ比統計値

Table 10. Odds ratio statistics for mixed cohorts

明らかに、これらのＳＮＰの多くは、両コホートにおいて非常に統計的に有意であった。これは、主に、本研究のための選択をもたらしたアプリオリな情報のせいであった。特に注意すべきは、フィッシャー値がｐ＜４．８１Ｅ−２０であって３（Ｔ／Ｔとして知られるＢＢ）であるＲＳ１０６１１７０であって、防御型の遺伝子型としての強力な可能性があることを示している。並列比較で、アイオワコホートとコロンビアコホートの間にはいくつかの違いがあることが明らかになった。

Clearly, many of these SNPs were very statistically significant in both cohorts. This was mainly due to a priori information that led to the choice for this study. Of particular note is RS1061170 with a Fisher value of p <4.81E-20 and 3 (BB known as T / T), which has a strong potential as a protective genotype Is shown. A side-by-side comparison revealed some differences between the Iowa and Columbia cohorts.

  In order to further determine which SNP / genotype is protective or contributing, the Fisher method was used as the basis for genotype penetration variance. To do this, the proportion of genotypes of case and control were calculated and the absolute value of their difference was calculated. Table 11 provides this information and is arranged in order of decreasing frequency.

  Table 11. Genotype penetration dispersion

仮定された防御モデル：本研究では、予備実験で、ＡＭＤから防御すると可能性があると思われるＳＮＰの組み合わせが示された。この仮説を検定するために、ＮＣＩの詳細な説明について手作りモデルを構築した。モデル図を図２Ａに示す。このモデルは、仮定（ＩＦ）文として以下のように記載されうる：
ＲＳ５４７１５４がＧ／Ａであり、かつＲＳ１０６１１７０がＴ／Ｔであるか、または
ＲＳ５４７１５４がＧ／Ａであり、かつＲＳ１０６１１７０がＣ／Ｃであるか、または
ＲＳ４１５１６６７がＴ／Ａであり、かつＲＳ１０６１１７０がＣ／Ｔであるか、または
ＲＳ４１５１６６７がＴ／Ａであり、かつＲＳ１０６１１７０がＣ／Ｃである場合には、
この人物は、ＡＭＤから防御されている。

Hypothetical model of protection: In this study, preliminary experiments showed combinations of SNPs that could potentially protect against AMD. To test this hypothesis, a handmade model was built for a detailed explanation of NCI. A model diagram is shown in FIG. 2A. This model can be described as an assumption (IF) statement as follows:
RS547154 is G / A and RS1061170 is T / T or RS547154 is G / A and RS1061170 is C / C or RS4151667 is T / A and RS1061170 is C / T or RS4151667 is T / A and RS1061170 is C / C
This person is protected from AMD.

Thus, this model gives the following four combinations of genotypes that may protect against AMD (the combination that makes the model “true”):
1. RS547147 which is G / A and RS1061170 which is T / T
Control person 8.82%, case person 5.45%
2. RS547147 which is G / A and RS1061170 which is C / C
Control person 7.22%, case person 1.93%
3. RS4151667 which is T / A and RS1061170 which is C / T
Control person 4.8%, case person 2.02%
4). RS4151667 which is T / A and RS1061170 which is C / C
Control person 3.47%, case person. 79%.

When this model was applied to the samples, the following results were obtained for a mixed cohort of Iowa and Colombia:
• 794 cases had no protective factors (model was false). . . 90.12%
• 88 controls had protective factors (model was true). . . 23.52%
・ 87 patients had protective factors. . . 9.88%
• 286 controls did not have protective factors. . . 76.47
Note: Statistics for all models were applied to the mixed cohort and looked at their true positive (TP), false negative (FN), false positive (FP), and true negative (TN) percentages. . These numbers were then entered into a 2 × 2 table where all statistics were generated. Table 12 shows the statistics for each cohort.

遺伝的アルゴリズム（ＧＡ）から得られたモデル：ＧＡモジュールがＳＮＰのより良好な組み合わせを見つけることができたか否かを見ようとして、ＧＡモジュールを作動させて、反転データに対するモデルを学習させた（防御型モデルを学習させるため）。以下のものを含む、さまざまなパラメータ設定値を利用した：
・モデルタイプ：該モデルが、「かつ（ＡＮＤ）」および「または（ＯＲ）」の組み合わせ、および「ＡＮＤ」だけ、または「ＯＲ」だけをもつか否かを示している。
・モデルサイズ：いくつのＳＮＰがモデルの中に存在しうるかについての上限値を示す。
・ＧＡ特異的パラメータ：例えば、世代、モデル数など。 Table 12. Defense model statistics assumed by NCI

Model obtained from the genetic algorithm (GA): In order to see if the GA module was able to find a better combination of SNPs, the GA module was activated to learn the model for inversion data (protection) To learn the type model). Various parameter settings were used, including:
Model type: indicates whether the model has a combination of “and (AND)” and “or (OR)” and only “AND” or “OR”.
Model size: indicates the upper limit on how many SNPs can exist in the model.
GA-specific parameters: for example, generation, number of models, etc.

  Generally speaking, a model consisting only of a small-scale AND is preferable. There are two reasons for this. First, in a model consisting only of ANDs, the interpretation is unambiguous because all of its SNPs are required to be true for a model that should be true, but in a model with OR, it is true. Since it is not required that all SNPs exist for a model to be, there is a certain level of uncertainty introduced. Second, since the number of SNPs to be measured decreases, the smaller the model, the easier the interpretation.

  Exemplar uses a two-thirds-one-third validation method to avoid over-learning the input data with the desired outcome that results in more generally applicable results.

  Furthermore, since there were two separate experimental groups, it was possible to build a model based solely on Colombian data and test the resulting model against the Iowa Cohort. If the performance of this model is consistent across the two groups, the general applicability of this model is strongly demonstrated. This is particularly interesting given the interesting statistical differences between the two groups, as discussed in the statistics section above. Given these mutations, GA can find a good fit model for the Colombian data, but the Iowa data may not perform well.

  GA found models that performed well for the entire Colombian, Iowa, and mixed-group data sets. As the model was trained on Colombian data, the model's performance on Colombia data was better than Iowa data as expected. Nevertheless, the performance of this model is noteworthy given that GA had no prior knowledge of the Iowa data and there were significant statistical differences between the main SNPs between the two cohorts. is there. The optimal model obtained performed better than the model handmade and assumed for the mixed cohort (RS1061170). First, this model included another section, “INSELECTT is homozygous and (AND) RS547154 is GG”, but further investigation revealed that this section was not related to model interpretation. Since it was judged that it was, this was excluded and the model with the same grade was created. The final model diagram can be seen in FIG. 2C.

The GA specific options for this task were as follows:
Models: 1500-This is the number of models built internally by GA as the basis for developing a new generation of models;
Iterations: 25—This is the number of evolutionary iterations the model has been considered before finding a solution;
Model size: 5-This is the size that allows the model to appear up to 16 genotypes in a single model;
Model type: AND / OR type—This allows GA to build a model that can use both types and / or types.

This model can be written in the hypothesis as follows:
If RS1048709 is G / G and RS1061170 is T / T, or RS547154 is G / A, or RS4151667 is T / A, or INDELTT is + / +
This person is protected from AMD.

This model gives the following four distinct genotypes or combinations of genotypes that protect against AMD (a combination that makes the model “true”):
1. RS1048709 is G / G, and RS1061170 is T / T
-Present in 14.20% of cases and 34.31% of controls. RS547154 is G / A
• Present in 9.6% of cases and 18.32% of controls. RS4151667 is T / A
• Present in 4.2% of cases and 9.92% of controls. INSELECT is + / +
Present in 1.45% of cases, 6.86% of controls Applying this model to the sample resulted in the following for a mixed cohort of Iowa and Colombia:
-682 cases (74.78%) had no protective factors and 230.

      • 204 controls (55.74%) had protective factors and 162 did not.

  Table 13 shows the statistics for each cohort. GA performed well across the board. Overall, those with the protective factor described by this model were 3.66581 times less likely to be AMD than those without the protective factor.

  Table 13. Model statistics obtained from genetic algorithms

この２つのコホート間におけるいくつかの主要ＳＮＰには明確な統計学的差異があることを考えると、より正確に転帰／防御を予測する単一のモデルを発見することがヒトにとっても、機械学習にとっても同じように難問であった。手作りモデルは、立派な成績を上げ、興味深いことに、ＳＮＰをＯＲで同一にまとめるなど、ＧＡが同定したのと同じＳＮＰのヘテロ接合的組み合わせを同定した（ＡＢであるＲＳ５４７１５４、ＡＢであるＲＳ４１５１６６７）。

Given that there are clear statistical differences in some of the major SNPs between the two cohorts, finding a single model that more accurately predicts outcome / protection can be machine learning for humans as well. It was equally difficult for me. The handmade model identified excellent results and, interestingly, identified heterozygous combinations of the same SNPs that GA identified, such as putting SNPs together in OR (AB RS547154, AB RS4151667). .

  Despite the difficulty of this task, GA has performed well for an out of sample test (Fischer value Iowa p <0.0000749). GA was superior to handmade models for all cohorts. Nevertheless, the handmade model does the job of predicting the results very well. Other modified models of this model were tested but could not improve their performance. Given that there are many possibilities for SNP / genotype / logical operator combinations, this is expected, so that tens of thousands of model variations can be tested within an appropriate time frame. The value of the machine learning method that can be performed.

  Given the high statistical significance of a single SNP (T / T RS1061170: × 2 = 97.25), one might conclude that it alone can predict the risk of AMD. To test whether a single SNP model or multilocus model might be suitable for predicting protection in the general population, a permutation test was performed on the data. A permutation test shows that a single SNP has a chi-square score of 4.8153 for 3000 permutations with a random mix of data, rather than either a GA or handmade model. In contrast, GA was 3.3157, and the handmade model was 1.2207, indicating a much higher probability of producing statistically significant results. With respect to odds ratio evaluation, the single SNP had OR> 1.5 and 625 reordering, while the GA model was 133 and the handmade model was 46. This handmade model simply represents a combination of genotypes that rarely exists in any sample. Eventually, the GA model showed the most correct case-control results and sort results.

A comprehensive look at model statistics, ROC plots, and permutation tests may indicate that the multilocus model method for predicting outcome is superior to a single locus across diverse groups .
CONCLUSION In conclusion, this study broadly illustrates and refines the role of the second complement pathway in the pathobiology of AMD, and suggests that infection and / or inflammation play a major role in this universal disease The model is further enhanced (Hageman et al., 2005, supra; Anderson et al., 2002, supra; Hageman et al., 2001, supra).

Example 3 Administration of protective BF protein or C2 protein to prevent the development of AMD The subject has signs and / or symptoms of AMD, including drusen. This subject has been determined to be negative for the protective polymorphisms, R32Q and L9H in BF, and IVS 10 and E318D in C2. This subject is recommended to be treated with protective BF protein (having R32Q polymorphism). To this subject, a sufficient amount of protective BF is placed in saline to give a serum concentration of BF of 9 to 31 mg / dL and administered intravenously once a month for 6 months. At this time, the subject is observed for drusen and other signs and / or symptoms of AMD. If the signs and / or symptoms of AMD have not progressed, do the protective BF once a month at the indicated frequency, but at least once every six months, observing the patient's clinical status Continue indefinitely.

  In another clinical dosing regimen, protective BF protein is administered intranasally once a day to more continuously expose the protective effect of the protein.

  All publications and patent applications mentioned in the specification are indicative of the level of skill in the technical field to which this invention pertains. All publications and patent applications are incorporated herein by reference as if each publication and patent application were specifically and individually described as being incorporated herein.

  In view of the many possible embodiments in which the principles of the present invention can be utilized, it is to be appreciated that the illustrated embodiments are only preferred examples of the present invention and are within the scope of the present invention. Should not be considered as limiting. Rather, the scope of the present invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

It is the schematic of SNP in BF and C2, and a haplotype analysis result. The SNPs used in this study are shown with predicted haplotypes, odds ratio (OR) P values (P), and combined (CAS) and control (CON) frequencies. The 95% confidence interval for H7 is (0.33-0.61) and for H10 is (0.23-0.56). The haplotype of the ancestor (chimpanzee) is represented as Anc. Haplotype H2 (available on NCBI accession number: AL662849 on February 8, 2006), haplotype H5 (used on NCBI accession number: AL645922 and NCBI accession number: NG_004658 on February 8, 2006) ), And haplotype H7 (NCBI accession number: NG — 000013 available on February 8, 2006) have been sequenced and both C2 and BF genes have been sequenced. There are no other non-synonymous mutants (Stewart et al. (2004) Genome Res. 14: 1176-87). Shows combinatorial complementary gene analysis. Individual SNP analysis has revealed that several combinations of SNPs are possible that protect individuals from developing AMD. To test these, an empirical model was first applied. FIG. 2A shows a model diagram that is interpreted as a combination of four genotypes that may protect against AMD. (1) rs641153 (R32Q) is G / A, and rsl061170 (Y402H) is C / T; (2) rs547154 is G / A, and rsl061170 is C / C; / A and rsl061170 are C / T; (4) rs4161667 is T / A and rsl061170 is C / C. Applying these models resulted in the distribution shown in FIG. 2B for each of the Iowa, Columbia, and mixed cohorts. Fisher's exact test was performed on these distributions and proved that the p-values were P = 0.00237, P = 4.28 × 10 ⁻⁸ , and P = 7.90 × 10 ⁻¹⁰ . . For comparison, an exemplary software, using a machine learning method known as a genetic algorithm, created a defense model that gave the “best fit” to the data. The resulting best model is shown in FIG. 2C. This model describes the following four possible genotypes or combinations of genotypes that protect against AMD: the combination that makes the model “true”. These genotypes are: (1) rs1048709 (R150R) is G / G, and rs1061170 is C / C; or (2) rs547154 is G / A; or (3) rs4151667 is T / A; or (4) CFH The intron 2 mutant is delTT. Model performance is shown in FIG. 2D for the Iowa, Columbia, and mixed cohorts. These distributions proved that the p-values were P = 7.49 × 10 ⁻⁵ , P = 2.97 × 10 ⁻²² , and P = 1.69 × 10 ⁻²³ , respectively. Shows combinatorial complementary gene analysis. Individual SNP analysis has revealed that several combinations of SNPs are possible that protect individuals from developing AMD. To test these, an empirical model was first applied. FIG. 2A shows a model diagram that is interpreted as a combination of four genotypes that may protect against AMD. (1) rs641153 (R32Q) is G / A, and rsl061170 (Y402H) is C / T; (2) rs547154 is G / A, and rsl061170 is C / C; / A and rsl061170 are C / T; (4) rs4161667 is T / A and rsl061170 is C / C. Applying these models resulted in the distribution shown in FIG. 2B for each of the Iowa, Columbia, and mixed cohorts. Fisher's exact test was performed on these distributions and proved that the p-values were P = 0.00237, P = 4.28 × 10 ⁻⁸ , and P = 7.90 × 10 ⁻¹⁰ . . For comparison, an exemplary software, using a machine learning method known as a genetic algorithm, created a defense model that gave the “best fit” to the data. The resulting best model is shown in FIG. 2C. This model describes the following four possible genotypes or combinations of genotypes that protect against AMD: the combination that makes the model “true”. These genotypes are: (1) rs1048709 (R150R) is G / G, and rs1061170 is C / C; or (2) rs547154 is G / A; or (3) rs4151667 is T / A; or (4) CFH The intron 2 mutant is delTT. Model performance is shown in FIG. 2D for the Iowa, Columbia, and mixed cohorts. These distributions proved that the p-values were P = 7.49 × 10 ⁻⁵ , P = 2.97 × 10 ⁻²² , and P = 1.69 × 10 ⁻²³ , respectively. Shows combinatorial complementary gene analysis. Individual SNP analysis has revealed that several combinations of SNPs are possible that protect individuals from developing AMD. To test these, an empirical model was first applied. FIG. 2A shows a model diagram that is interpreted as a combination of four genotypes that may protect against AMD. (1) rs641153 (R32Q) is G / A, and rsl061170 (Y402H) is C / T; (2) rs547154 is G / A, and rsl061170 is C / C; / A and rsl061170 are C / T; (4) rs4161667 is T / A and rsl061170 is C / C. Applying these models resulted in the distribution shown in FIG. 2B for each of the Iowa, Columbia, and mixed cohorts. Fisher's exact test was performed on these distributions and proved that the p-values were P = 0.00237, P = 4.28 × 10 ⁻⁸ , and P = 7.90 × 10 ⁻¹⁰ . . For comparison, an exemplary software, using a machine learning method known as a genetic algorithm, created a defense model that gave the “best fit” to the data. The resulting best model is shown in FIG. 2C. This model describes the following four possible genotypes or combinations of genotypes that protect against AMD: the combination that makes the model “true”. These genotypes are: (1) rs1048709 (R150R) is G / G, and rs1061170 is C / C; or (2) rs547154 is G / A; or (3) rs4151667 is T / A; or (4) CFH The intron 2 mutant is delTT. Model performance is shown in FIG. 2D for the Iowa, Columbia, and mixed cohorts. These distributions proved that the p-values were P = 7.49 × 10 ⁻⁵ , P = 2.97 × 10 ⁻²² , and P = 1.69 × 10 ⁻²³ , respectively. Shows combinatorial complementary gene analysis. Individual SNP analysis has revealed that several combinations of SNPs are possible that protect individuals from developing AMD. To test these, an empirical model was first applied. FIG. 2A shows a model diagram that is interpreted as a combination of four genotypes that may protect against AMD. (1) rs641153 (R32Q) is G / A, and rsl061170 (Y402H) is C / T; (2) rs547154 is G / A, and rsl061170 is C / C; / A and rsl061170 are C / T; (4) rs4161667 is T / A and rsl061170 is C / C. Applying these models resulted in the distribution shown in FIG. 2B for each of the Iowa, Columbia, and mixed cohorts. Fisher's exact test was performed on these distributions and proved that the p-values were P = 0.00237, P = 4.28 × 10 ⁻⁸ , and P = 7.90 × 10 ⁻¹⁰ . . For comparison, an exemplary software, using a machine learning method known as a genetic algorithm, created a defense model that gave the “best fit” to the data. The resulting best model is shown in FIG. 2C. This model describes the following four possible genotypes or combinations of genotypes that protect against AMD: the combination that makes the model “true”. These genotypes are: (1) rs1048709 (R150R) is G / G, and rs1061170 is C / C; or (2) rs547154 is G / A; or (3) rs4151667 is T / A; or (4) CFH The intron 2 mutant is delTT. Model performance is shown in FIG. 2D for the Iowa, Columbia, and mixed cohorts. These distributions proved that the p-values were P = 7.49 × 10 ⁻⁵ , P = 2.97 × 10 ⁻²² , and P = 1.69 × 10 ⁻²³ , respectively. BF along the retinal pigment epithelium (RPE) -choroid (CH) complex in a section obtained from an unfixed eye of a 72-year-old elderly donor with early stage AMD (FIG. 3A) ), Ba (fragment of full-length factor B) (FIG. 3B), and C3 (FIG. 3C). Anti-BF antibodies (Quidel; reaction product is red) specifically label drusen (D) along their periphery and Bruch's membrane and choroidal stroma. Anti-Ba antibody (Quidel; reaction product is purple) labels Bruch membranes and RPE-associated patches. Note that the distribution of BF is similar to the distribution of C3. The brown coloration in the cytoplasm and choroid of RPE is due to melanin. Bruch's membrane (BM); retina (R).

Claims (21)

The possibility that age-related macular degeneration (AMD) risk of developing or has progressed to a method to assist in assessing in a human subject, the subject is, following a) to d) of A method comprising determining whether to have one or more:
a) A or G at rs641153 of the complement factor B (BF) gene, or R or Q at position 32 of the BF protein;
b) A or T at rs4151667 of the BF gene, or L or H at position 9 of the BF protein;
c) C2 G or T in rs547154 gene; and d) said C2 C or G at rs9332 73 9 gene, or E or D. In position 318 of the C2 protein The method of claim 1, wherein the determining comprises analyzing a biological sample obtained from the human subject. The method according to claim 1 or 2 , further comprising the step of determining whether the subject has one or more of the following a) to b):
a) Complement factor H (CFH) Contact Keru 2 base deletion (SEQ ID NO: gene 6); and b) C or T at rs1061170 the CFH gene, or in position 402 of the CFH protein, Y or H. The method according to any one of claims 1 to 3, wherein the subject is asymptomatic for macular degeneration. The method according to any one of claims 1 to 3 , wherein the subject has symptoms of macular degeneration. The method of claim 2 , wherein the sample is an available body fluid. 3. A method according to claim 1 or 2 , comprising detecting a genotype from the cell of interest. 3. The method of claim 1 or 2 , comprising detecting a protein variant in the subject. And detecting the mRNA from the cells of the subject, according to claim 1 or 2 wherein. 3. The method of claim 1 or 2 , further comprising determining whether the subject is homozygous or heterozygous for the polymorphism. A kit for assessing in human subjects the risk of developing age-related macular degeneration (AMD) or the likelihood that the disease has progressed, wherein one or more of the following polymorphisms or A kit comprising reagents for detecting one or more allelic variants:
a) A or G at rs641153 of the complement factor B (BF) gene, or R or Q at position 32 of the BF protein;
b) A or T at rs4151667 of the BF gene, or L or H at position 9 of the BF protein;
c) C2 G or T in rs547154 gene; and d) said C2 C or G at rs9332 73 9 gene, or E or D. In position 318 of the C2 protein Comprising reagents for detecting one or more of the following polymorphisms or one or more allelic variants in a sample obtained from said subject, according to claim 1 1, wherein the kit:
a) Complement factor H (CFH) Contact Keru 2 base deletion (SEQ ID NO: gene 6); and b) C or T at rs1061170 the CFH gene, or in position 402 of the CFH protein, Y or H. Comprising reagents for detecting two or more polymorphisms or more allelic variants claim 1 1 wherein kits. Comprising an oligonucleotide for detecting the polymorphism, claim 1 1 wherein kits. Further comprising, according to claim 1 1, wherein the kit of reagents for amplifying a target polynucleotide sequence comprising the polymorphism. Including immobilized oligonucleotides on a solid support, according to claim 1 1, wherein kits. Contain specific binding proteins that specifically recognize and bind the allelic variant, according to claim 1 1, wherein kits. Age-related macular comprising an oligonucleotide probe capable of hybridizing under stringent conditions to one or more nucleic acid molecules having a protective polymorphism selected from the group consisting of: A microarray for assessing in human subjects the risk of developing degeneration (AMD) or the likelihood that the disease has progressed :
a) R32Q polymorphism in BF (rs641153);
b) L9H polymorphism in BF (rs4151667);
c) IVS 10 polymorphism in C2 (rs547154); and d) E318D polymorphism in C2 (rs93332739). Under stringent conditions, further comprising the following a) to c) oligonucleotide probes capable of hybridizing to one or more nucleic acid molecules having a polymorphism selected from the group consisting of, claims 1 to 8, wherein Microarray:
a) deletion of 2 bases in CFH (SEQ ID NO: 6) polymorphism;
b) R150R polymorphism in BF; and c) Y402H polymorphism in CFH. An apparatus for assessing in human subjects the risk of developing age-related macular degeneration (AMD) or the likelihood of progression of the disease, under stringent conditions: a) to d A device comprising a microarray comprising oligonucleotide probes capable of hybridizing to one or more nucleic acid molecules having a protective polymorphism selected from the group consisting of:
  a) R32Q polymorphism in BF (rs641153);
  b) L9H polymorphism in BF (rs4151667);
  c) IVS 10 polymorphism in C2 (rs547154); and
  d) E318D polymorphism in C2 (rs93332739). 21. The method according to claim 20, further comprising an oligonucleotide probe capable of hybridizing under stringent conditions to one or more nucleic acid molecules having a polymorphism selected from the group consisting of: apparatus:
  a) deletion of 2 bases in CFH (SEQ ID NO: 6) polymorphism;
  b) the R150R polymorphism in BF; and
  c) Y402H polymorphism in CFH.

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