MX2008004816A - Genes associated with macular degeneration - Google Patents

Abstract

Identification of variant genes correlated with age related macular degeneration, such as variant LOC387715, variant SYNPR and variant PDGFC;methods of identifying or aiding in identifying individuals at risk for developing age related macular degeneration.

Description

GENES ASSOCIATED WITH MACULAR DEGENERATION PROVIDING FUNDS This invention was made with the support of the government of the States United under Grant Number HG000060 and Grant Number EY015771, from the National Institutes of Health The United States government has certain rights over the invention CROSS REFERENCE TO THE RELATED APPLICATION This application claims the benefit of the Provisional Application of E U A No 60 / 726,061, filed on October 11, 2005 The teachings of this provisional application referred to, are incorporated herein by reference in their entirety BACKGROUND OF THE INVENTION Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Its incidence is increasing as the time of life lengthens and the older population expands (D S Fpedman et al, Arch Ophthalmol 122, 564 (2004)) is a chronic disease characterized by a progressive destruction of the central region of the retina (macula), causing the loss of central field vision (J. Tuo, CM Bojanowski, CC Chan, Prog Retin Eye Res 23, 229 (2004)). A key feature of AMD is the formation of extracellular deposits called druse that are concentrated in and around the macula behind the retina between the retinal pigment epithelium (RPE) and the choroid. The risk of developing AMD is determined by the complex interplay of genetic variants, many of which are still unidentified. Additional information on the genetic determinants of AMD would be very valuable to the field.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the identification of variations in human genes that are correlated with a predisposition to AMD. Such variations and the variant genes in which they occur are useful in identifying or assisting in the identification of individuals at risk of developing AMD, as well as for diagnosis or assisting in the diagnosis of AMD. The invention also relates to methods for identifying or helping to identify individuals at risk of developing AMD, methods to diagnose or aid in the diagnosis of AMD, polynucleotides (e.g., probes, primers) useful in methods, equipment Diagnostics containing probes or primers, methods for treating an individual at risk for, or suffering from AMD and compositions useful for the treatment of an individual at risk for, or suffering from, AMD. The Requesters analyzed SNPs throughout the genome, genotyping data from individuals with AMD and individuals without AMD (controls) and looked for simple associations in LOC387715 and other sites that appear to interact. A variant known to play a role in the risk of developing AMD is found in the LOC387715 gene. As described herein, the Requesters formed an association between LOC387115 and AMD. In addition, they provided evidence that the interaction of the variants in the LOC387715 genes, the sinaptoporin (SYNPR), and the platelet derived growth factor C (PDGFC), all of which are located in the known AMD binding peaks. , contribute to a susceptibility for AMD. These interactions, together with the complement factor H (CFH) association that the Requesters previously identified, appear to be a significant genetic risk for AMD. In one embodiment, the present invention provides useful polynucleotides for detection or aiding in the detection of a gene LOC387715, which is correlated with the appearance of AMD in humans, a SYNPR gene that is correlated with the appearance of AMD in humans and a PDGFC gene that is correlated with the appearance of AMD in humans. The phrases "correlated with the appearance of AMD in humans" and "correlated with the appearance of AMD" are used interchangeably in the present. In the specific modalities, the invention relates to polynucleotides useful for the detection or aid in the detection of variations in each gene that are correlated with AMD in humans. In another embodiment, the present invention provides methods and compositions useful for identifying or helping to identify individuals at risk of developing AMD. In a further embodiment, the methods and compositions of the invention can be used for the treatment of an individual suffering from AMD or at risk of developing AMD. Also, the subject of the invention are diagnostic kits for detecting a variant LOC387715 gene, a variant SYNPR gene and / or a variant PDGFC gene, alone or in combination, in a sample from an individual. Such equipment can also be useful to detect a variant CFH gene "; which comprises a variation in the CFH gene that is correlated with the appearance of AMD. Such equipment is useful to identify or help identify individuals at risk of developing AMD, as well as to diagnose or assist in the diagnosis of AMD in an individual. In specific embodiments, the invention provides isolated polynucleotides for the detection of a variant LOC387715 gene; isolated polynucleotides for the detection of a variant SYNPR gene; and isolated polynucleotides for the detection of a variant PDGFC gene. The isolated polynucleotide comprises a nucleic acid molecule that specifically detects a variation in the LOC387715 gene that is correlated with AMD in humans; a variation in the SYNPR gene that is correlated with AMD in humans; or a variation in the PDGFC gene that is correlated with the appearance of AMD in humans. The isolated polynucleotides are useful for detecting, in a sample from an individual, a variant LOC387715 gene, a variant SYNPR gene or a variant PDGFC gene that is correlated with AMD in humans. The work described here provides strong evidence of genetic interactions at three sites on different chromosomes for AMD susceptibility. The LOC387715 variants work in conjunction with the synaptophorin variants (SYNPR) and the platelet derived growth factor C (PDGFC) variants. In contrast, the CFH variants independently contribute to the genetic risk of an individual to develop AMD. The estimated PAR reached a level (0.55 to 0.71) that is as high as at the previously estimated level (0.46 to 0.71) of the genetic contribution to the AMD (16), which supports the hypothesis that the reported genetic networks capture a portion of genetic risk for AMD, for example, in populations of European descent. The contribution of these genetic networks for susceptibility to AMD in other populations can be confirmed or determined using the methods described herein.

DETAILED DESCRIPTION OF THE INVENTION General As described herein, the Applicants have investigated the LOC387715 site, independently and in conjunction with other genes, in the genome-wide association data obtained from the genotyping of individuals from the study of related eye disease. with age (AREDS) for more than 100,000 single nucleotide polymorphisms (SNP) (AREDS Research Group, Ophthamology 107, 2224 (2000)). As also described herein, the results of that investigation have shown that the association of three gene variants (LOC387715, sinaptoporin (SYNPR), and platelet derived growth factor C (PDGFC) with the development of the AMD and support the role of their interaction in the susceptibility to AMD The variants of the three genes are represented in the present, respectively, as vLOC387715, vSYNPR and vPDGFC These interactions, together with the association of complement factor H (CFH) that the Requesters previously identified seem to constitute a considerable genetic risk for AMD, the valuation of the variants of the three genes that are associated with the appearance of AMD and the variation of the variants of the three genes in combination with the variation of a variant CFH gene that is correlated with the appearance of the AMD, are useful in the identification or help in the identification of an individual in risk of developing AMD, as well as in diagnosis or aid in the diagnosis of AMD in an individual (for example, a human) Variations in the LOC387715 gene, variations in the SYNPR gene and variations in the PDGFC gene that showed be correlated (associated) with AMD in humans, are useful for the diagnosis and initial treatment of individuals predisposed to AMD The determination of the genetic makeup of the LOC387715 gene, the SYNPR gene, and the PDGFC gene in an individual (human ), is useful for treating AMD in early stages, or even before an individual shows any symptoms of AMD. In addition, diagnostic tests for the genotype LOC387715, SYNPR and PDGFC, may allow individuals, such as those who they are at risk to develop AMD, alter their behavior to reduce the environmental risks that contribute to the development of AMD (for example, smoking) and, as a result, reduce their risk of e develop AMD, reduce the severity of AMD and / or delay its onset In one embodiment, the present invention relates to the identification of the vLOC387715 gene, the vSYNPR gene and the vPDGFC gene, which are correlated with the appearance of AMD , a predisposition for (increased probability of developing) AMD in humans These variants are useful to identify or help identify individuals in need of developing AMD, as well as to diagnose or assist in the diagnosis of AMD. The invention is also relates to methods for identifying or helping to identify individuals at risk of developing AMD, methods and compositions for detecting such variations that predispose a human to AMD, methods to diagnose or aid in the diagnosis of AMD, polynucleotides (e.g. , probes, primers) useful in the methods, diagnostic equipment that contains probes or primers and that are useful in the methods of this invention, methods for treating an individual at risk for, or suffering from, AMD and compositions useful for treating an individual at risk for, or suffering from, AMD. The variants of the three genes that showed in the present that they interact, can be evaluated in the methods of the present invention alone (without the valuation of other factors, such as without the variation of a variant CFH gene that is correlated with the appearance of the AMD), or in combination with the assessment of additional factors, such as in combination with the valuation of a variant CFH gene that is correlated with AMD or clinical assessment. The LOC387715, SYNPR and PDGFC genes can be the cDNA and the genomic form of the gene, which can include the upstream and downstream regulatory sequences. See, for example, the entry of the LOC387715 gene from homosapiens at http://www.ncbi.nlm.nih.gov; synaptophorin (rat protein P22831 EMBL and synaptorin SYNPR, Gene ID 66030 Entrez Gene at http://rat.embl.de: SYNPR_MOUSE Q8BGN8 at http://us.expasv.org; human protein Q8BGN9-Sinaptoporina EMBL and sinaptoporina SYNPR [Homosapiens] at http://harvester.embl.de); PDGFC (access to Genbank AF336376; Utela et al Circulation 2001; 103: 2242-2247), for example sequences, which are not intended to be limiting in any way. The polynucleotide probes and primers of the invention can hybridize to any contiguous portion of one of the three genes (LOC387715, SNYPR or PDGFC or to any contiguous portion of one of the three gene variants (vLOC387715, vSNYPR or vPDGFC). SYNPR, and PDGFC may further include localized sequences adjacent to the coding region at both 5 'and 3' ends for a distance of about 1-2 kb at either end, such that the gene corresponds to the length of the full-length mRNA. Sequences which are located at the 5 'end of the coding region and which are present in the mRNA are referred to as the 5"untranslated sequences. The sequences which are located at the 3' end or downstream of the coding region and which are present in the mRNA are referred to as the 3 'untranslated sequences.The subject of the invention are also the isolated vLOC387715 polypeptides; the vSYNPR polypeptides isolated and the isolated vPDGFC polypeptide and its use in the methods of the present invention, such as methods for identifying or helping to identify individuals at risk of developing AMD, methods for detecting such variations that predispose a human to AMD, and methods for To diagnose or aid in the diagnosis of AMD, polypeptide sequences vLOC387715 include human polypeptide sequences, such as the Ala69Ser change encoded by the coding change in the LOC387715 gene described by Fisher et al. (8) and non-human polypeptide sequences ( for example, rat, mouse). Similarly, vSYNPR polypeptides and vPDGFC polypeptides include human and non-human sequences. The LOC387715, SYNPR and PDGFC polypeptides can be encoded by a full-length coding sequence or by any portion of the coding sequence and the vLOC387715 polypeptides, vSYNPR and vPDGFC can be encoded by a full-length coding sequence or by any portion of the coding sequence, provided that the encoded polypeptide has the desired functional activity or property (eg, enzymatic activity, binding to a ligand, signal transduction).

Probes and primers of the LOC387715, SYNPR and PDGFC polynucleotides In certain embodiments, the invention provides isolated and / or recombinant polynucleotides that specifically detect a variation in the LOC387715 gene that is correlated with the onset of AMD, a variation in the gene SYNPR that is correlated with the appearance of AMD, or a variation in the PDGFC gene that is correlated with the occurrence of AMD or a combination thereof. The polynucleotide probes of the invention hybridize to a variation (referred to as a variation of interest) in such a LOC387715 gene, SYNPR gene or PDGFC gene, and the flanking sequence, in a specific manner, and typically, thus have a sequence that is total or partially complementary to the sequence of the variation and the flanking region. The polynucleotide probes of the invention can hybridize to a segment of a gene or to a DNA comprising a variation of interest, such that the variation is aligned with a central portion of the probe or with another portion of the probe, such as a portion of the probe. probe terminal. In one embodiment, an isolated polynucleotide probe of the invention hybridizes, under stringent conditions, to a nucleic acid molecule comprising a LOC387715 variant gene that is correlated with AMD, a variant SYNPR gene that is correlated with the appearance of AMD , or a variant PDGFC gene that is correlated with the appearance of AMD in humans, or a portion or allelic variant thereof. In another embodiment, an isolated polynucleotide probe of the invention hybridizes, under stringent conditions, to a nucleic acid molecule comprising at least 10 contiguous nucleotides of a LOC387715 gene, a SYNPR gene, a PDGFC gene, a variant LOC387715 gene that is correlated with AMD, a variant SYNPR gene that is correlated with AMD, a variant PDGFC gene that is correlated with AMD or an allelic variant thereof, wherein the nucleic acid molecule comprises a variation that is correlated with the occurrence of AMD in humans. In certain embodiments, a polynucleotide probe of the invention is a probe specific for the allele. The design and use of the probes specific for the allele to analyze the polymorphisms is described in, for example, Saiki et al., Nature 324: 163-166 (1986); Dattagupta, EP 235726; and Saiki WO 89/1 1548. Specific allele probes can be designed to hybridize to a segment of a target DNA of an individual, but not to hybridize to the corresponding segment of another individual, due to the presence of different polymorphic forms or variations in the respective segments of the two individuals. Hybridization conditions must be sufficiently stringent, so that there is a significant difference in the intensity of hybridization between the alleles. In some embodiments, a probe hybridizes to only one of the alleles. A variety of variations in the LOC387715 gene, the SYNPR gene, and the PDGFC gene or any combination of such variations that predisposes an individual to AMD can be detected by the methods and polynucleotides described herein. For example, any nucleotide polymorphism of a coding region, exon, boundary exon-intron, signal peptide, prime untranslated region, promoter region, enhancer sequence, unprimed 3 prime region or intron that is associated with AMD in humans it can be detected. These polymorphisms include, but are not limited to, changes that: alter the amino acid sequence of the proteins encoded by the LOC387715 gene, the SYNPR gene, and / or the PDGFC gene, produce alternative splicing products, create truncated products, introduce a codon of premature arrest, introduce a cryptic exon, alter the degree or expression to a greater or lesser degree, alter the tissue specificity of gene expression, introduce changes in the tertiary structure of the proteins encoded by LOC387715, SYNPR or PDGFC, introduce changes in the binding affinity or specificity of the proteins expressed by LOC387715, SYNPR or PDGFC or alter the function of the proteins encoded by LOC387715, SYNPR or PDGFC In a specific modality, the variation in the LOC387715 gene encodes an amino acid other than alanine (eg, sepna) at position 69 of the protein LOC387715 Other variant genes, such as those in which the variation is in a coding region (eg, variations encoding an amino acid other than the amino acid present in the corresponding position in a LOC387715 gene that is not correlated with the AMD, in the corresponding position in a SYNPR gene that is not correlated with the AMD or in a PDGFC gene that is not correlated with the AMD)), can be detected using the methods and compositions described herein In alternate fashion, the variant genes in which the variation is in a region not encoding can be detected using the methods and compositions described herein. It is further understood that the subject nucleotides include the polynucleotides which are variants of the nucleotides described herein, with the proviso that the vanishing polynucleotides maintain their ability to detect Specifically, a variation in the LOC387715 gene, the SYNPR gene or the PDGFC gene that is correlated with the appearance of AMD. Vanishing polynucleotides may include, for example, sequences that differ by one or more nucleotide substitutions, additions or deletions. , the isolated polynucleotide is a probe that hybridizes, under stringent conditions, to a variation in the LOC387715 gene that is correlated with the occurrence of AMD in humans, a variation in the SYNPR gene that is correlated with the onset of AMD in human, or a variation in the PDGFC gene that is correlated with the ap ary of AMD in humans. The term "probe" refers to a polynucleotide that is capable of hybridizing to another nucleic acid of interest. The polynucleotide can be natural, as in a purified restriction digestion, or it can be produced synthetically, recombinantly or by amplification of the nucleic acid (eg, PCR amplification). It is well known in the art how to perform hybridization experiments with nucleic acid molecules. The person with experience is familiar with the conditions of hybridization. Such hybridization conditions are referred to in standard textbooks, such as Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001); and in Current Protocols in Molecular Biology, eds. Ausubel et a! .. John Wíley & Sons (1992). Particularly useful in the methods of the present invention are polynucleotides that hybridize to a variation in the LOC387715 gene that is correlated with the occurrence of AMD in humans, a variation in the SYNPR gene that is correlated with the appearance of AMD in humans, or a variation in the PDGFC gene that is correlated with the occurrence of AMD in humans or a region of a LOC387715, SYNPR or variant PDGFC gene, under stringent conditions. Under stringent conditions, a polynucleotide that hybridizes to a LOC387715 variant gene, which is correlated with the occurrence of AMD in humans, a variant SYNPR gene, which is correlated with the occurrence of AMD in humans, or a variant PDGFC gene, which is correlated with the occurrence of AMD in humans, does not hybridize to the corresponding LOC387715 gene, SYNPR or PDGFC, which does not include the variation of interest. Hybridization of the nucleic acid is affected by conditions such as salt concentration, temperature, organic solvents, base composition, length of the complementary strands and the number of bad correspondences at the nucleotide base between the hybridizing nucleic acids, as will easily appreciate by those with ordinary skill in the art. Rigorous temperature conditions will generally include temperatures in excess of 30 ° C, or may be in excess of 37 ° C or 45 ° C. Rigor increases with temperature. For example, temperatures higher than 45 ° C are highly stringent conditions. The stringent conditions of the salt will ordinarily be less than 1000 mM, or may be less than 500 mM or 200 mM. For example, one could hybridize at 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a 2.0 x wash of SSC at 50 ° C. For example, the concentration of the salt in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 ° C to a high stringency of about 0.2 x SSC at 50 ° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. Both the temperature and the salt can be varied, or the temperature or concentration of the salt can be kept constant while the other variable changes. Particularly useful in the methods of the present invention are polynucleotides that are capable of hybridizing to a variant LOC387715 gene, which is correlated with the occurrence of AMD in humans, a variant SYNPR gene, which is correlated with the appearance of AMD in human, or a variant PDGFC gene, which is correlated with the occurrence of AMD in humans, or a region of a LOC387715 gene, SYNPR or PDGFC variant, under stringent conditions. It is understood, however, that the appropriate stringency conditions can be varied to encourage DNA hybridization. In certain embodiments, the polynucleotides of the present invention hybridize to a variant LOC387715 gene, which is correlated with the onset of AMD in humans, a variant SYNPR gene, which is correlated with the occurrence of AMD in humans or a PDGFC gene variant, which is correlated with the occurrence of AMD in humans, or a region of such variant LOC387715 gene, a variant SYNPR gene or a variant PDGFC gene, under highly stringent conditions. Under stringent conditions, a polynucleotide that hybridizes to a variation in the LOC387715 gene, a variation in the SYNPR gene or a variation in the PDGFC gene does not hybridize to the LOC387715, SYNPR or PDGFC gene that does not include the variation of interest. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6.0 x SSC at room temperature, followed by a 2.0 x SSC wash at room temperature. The combination of parametersHowever, it is much more important than the measurement of any single parameter. See, for example, Wetmur and Davidson, 1968. The sequences of the probe can also hybridize specifically to duplex DNA under certain conditions to form triple or higher order DNA complexes. The preparation of such probes and the suitable hybridization conditions is well known in the art. A polynucleotide probe or primer of the present invention can be labeled, so that it is detectable in a variety of detection systems, including, but not limited to, enzyme systems (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent , radioactive, chemical and luminescent. A polynucleotide probe or primer of the present invention may further include an extinguishing portion which when placed in proximity to a tag (eg, a fluorescent tag), causes there to be little or no signal from the tag. The detection of the tag can be done by direct or indirect means (for example, via a biotin / avidin or biotin / streptavidin link). It is not intended that the present invention be limited to any particular detection system or brand. In another embodiment, the isolated polynucleotide of the invention is a primer that hybridizes, under stringent conditions, adjacent, upstream or downstream to a variation in a LOC387715 gene, a SYNPR gene or a PDGFC gene that is correlated with the appearance of AMD in humans. The isolated polynucleotide can hybridize, under stringent conditions, to a nucleic acid molecule comprising all or a portion of a variant LOC387715 gene, variant SYNPR gene or variant PDGFC gene, which is correlated with the occurrence of AMD in humans. Alternatively, the isolated polynucleotide primer can hybridize, under stringent conditions, to a nucleic acid molecule comprising at least 50 contiguous nucleotides of a variant LOC387715 gene, variant SYNPR gene or variant PDGFC gene, which is correlated with the appearance of AMD in humans. For example, a polynucleotide primer of the invention can hybridize adjacent, upstream or downstream to the region of the LOC387715 gene encoding the amino acid 69 of the encoded protein. As used herein, the term "primer" refers to a polynucleotide that is capable of acting as a starting point for the synthesis of a nucleic acid, when placed under conditions in which the synthesis of a extension of the primer that is complementary to a strand of nucleic acid (eg, in the presence of nucleotides, an induction agent such as DNA polymerase and at suitable temperature, pH and electrolyte concentration). Alternatively, the primer may be capable of binding to a proximal nucleic acid when placed under conditions in which the ligation of two unbound nucleic acids occurs (e.g., in the presence of a proximal nucleic acid, an induction agent. such as DNA ligase and suitable temperature, pH and electrolyte concentration). A polynucleotide primer of the invention can be natural, as in a purified restriction digestion or it can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification, but alternatively, it can be double-stranded. If it is double-stranded, the primer is first treated to separate its strands before being used. Preferably, the primer is an oligodeoxyribonucleotide. The exact lengths of the primers will depend on many factors, including the temperature, source of the primer and the use of the method. In certain embodiments, the polynucleotide primer of the invention is at least 10 nucleotides long and hybridizes to one side or the other of a variation in the LOC387715, SYNPR or PDGFC gene that is correlated with the occurrence of AMD in humans. The object polynucleotides may contain alterations, such as one or more substitutions, additions or deletions of nucleotides, with the proviso that they hybridize to their LOC387715 gene, SYNPR and / or PDGFC target variant with substantially the same degree of specificity. In one embodiment, the invention provides a pair of primers that specifically detect a variation in the LOC387715 gene that is correlated with AMD, a variation in the SYNPR gene that is associated with AMD, or a variation in the PDGFC gene that is correlated with the appearance of AMD. In such a case, the first primer is hybridized upstream of the variation and a second primer is hybridized under the variation. For example, one of the primers hybridizes to a strand of a DNA region comprising a variation in the LOC387715 gene, a variation in the SYNPR gene that correlates with AMD or a variation in the PDGFC gene that is correlated with the appearance of the AMD, and the second primer hybridizes to the complementary strand of a DNA region comprising a variation in the LOC387715 gene that is correlated with the AMD, a variation in the SYNPR gene that is correlated with the AMD, or a variation in the PDGFC gene that is correlated with the appearance of AMD. As used herein, the term "DNA region" refers to a subchromosomal length of the DNA. In another embodiment, the invention provides an allele-specific primer that hybridizes to a site on the target DNA that overlaps a variation in the LOC387715 gene that is correlated with AMD, a variation in the SYNPR gene that is correlated with the AMD, or a variation in the PDGFC gene that is correlated with the appearance of AMD. A specific primer of the allele of the invention only primes the amplification of an allelic form with which the primer exhibits perfect complementarity. This primer can be used, for example, in conjunction with a second primer that hybridizes at a distal site. The amplification can then proceed from the two primers, resulting in a detectable product indicating the presence of a LOC387715 variant gene that is correlated with the onset of AMD, a variant SYNPR gene that is correlated with the occurrence of AMD, or a PDGFC gene variant that is correlated with the appearance of the AMD.

Detection assays In certain embodiments, the invention relates to polynucleotides useful for detecting a variation in a LOC387715, SYNPR or PDGFC gene, which is correlated with the onset of age-related macular degeneration. Preferably, these polynucleotides are capable of hybridizing, under stringent hybridization conditions, to a DNA region comprising a variation in the LOC387715 gene, a variation in the SYNPR gene, or a variation in the PDGFC gene that is correlated with the appearance of macular degeneration related to age. The polynucleotides of the invention can be used in any assay that allows detection of a variation in the LOC387715, SYNPR or PDGFC gene that is correlated with the onset of AMD. Such methods may include, for example, methods of DNA sequencing, hybridization, ligation or extension of the primer. In addition, any combination of these methods can be used in the invention. In one embodiment, the presence of a variation in the LOC387715 gene, SYNPR, PDGFC or combination thereof, which is correlated with the appearance of AMD is detected and / or determined by DNA sequencing. The determination of the DNA sequence can be carried out by standard methods such as dideoxy chain termination technology and gel electrophoresis, or by other methods such as pyrosequencing (Biotage AB, Uppsala, Suecia). For example, DNA sequencing by terminating the dideoxy chain can be performed using unlabeled primers and labeled terminators (eg, fluorescent or radioactive). Alternatively, sequencing can be performed using labeled primers and unlabeled terminators. The nucleic acid sequence of the DNA in the sample can be compared to the nucleic acid sequence of wild-type DNA or DNA that does not comprise a correlated variation with the appearance of AMD, to determine whether a variation in the LOC387715 gene is correlated with the AMD, a variation in the SYNPR gene that is correlated with AMD, a variation in the PDGFC gene that is correlated with the occurrence of AMD or a combination of such variations is present. In another embodiment, the presence of a variation in the LOC387715 gene that is correlated with the occurrence of AMD, a variation in the SYNPR gene that is correlated with the occurrence of AMD, a variation in the PDGFC gene that is correlated with the appearance of the AMD or a combination thereof, is detected and / or determined by hybridization. In one embodiment, a polynucleotide probe hybridizes to a variation in the LOC387715 gene, SYNPR gene or PDGFC gene, which is correlated with AMD and the flanking nucleotides, but not a LOC387715, SYNPR or PDGFC gene that does not contain a variation that It is correlated with the AMD. The polynucleotide probe can comprise nucleotides that are fluorescent, radioactive or chemical labeled to facilitate the detection of hybridization. Hybridization can be performed and detected by standard methods known in the art, such as by Northern blotting, Southern blotting, fluorescent in situ hybridization (FISH), or by hybridization to immobilized polynucleotides on a solid support, such as an array or microarray. of DNA. As used herein, the terms "DNA array" and "microarray" refer to an array of hybrid elements of the array. The elements of the array are arranged so that there is preferably at least one or more different array elements, immobilized on the surface of a substrate. The hybridization signal of each of the elements of the array is distinguishable individually. In another embodiment, the presence of a variation in the LOC387715 gene that is correlated with the appearance of the AMD is detected and / or determined by hybridization. In another modality, the presence of a variation in the SYNPR gene that is correlated with the appearance of AMD is detected and / or determined by hybridization. In another embodiment, the presence of a variation in the PDGFC gene that is correlated with the appearance of the AMD is detected and / or determined by hybridization. In a specific embodiment, the polynucleotide probe is used to hybridize genomic DNA by FISH. FISH can be used, for example, in metaphase cells, to detect a deletion in genomic DNA. The genomic DNA is denatured to separate the complementary strands inside the double helix structure of the DNA. The polynucleotide probe of the invention is then added to the denatured genomic DNA. If a variation in the LOC387715 gene that is correlated with the occurrence of AMD, a variation in the SYNPR gene that is correlated with the occurrence of AMD, or a variation in the PDGFC gene that is correlated with the occurrence of AMD is present, the probe will hybridize to the genomic DNA. The signal from the probe (eg, fluorescence) can then be detected through a fluorescent microscope for the absence of the signal. The absence of the signal, therefore, indicates the absence of a variation in the respective gene, which is correlated with the appearance of the AMD. In another specific embodiment, a labeled polynucleotide probe is applied to the immobilized polynucleotides in a DNA array. Hybridization can be detected, for example, by measuring the intensity of the labeled probe remaining in the DNA array after washing. The polynucleotides of the invention can also be used in commercial assays, such as in the Taqman assay (Applied Biosystems, Foster City, CA). In another modality, the presence of a variation in the gene LOC387715 that is elated with the occurrence of AMD, a variation in SYNPR that is elated with the occurrence of AMD, or a variation in the PDGFC gene that is elated with the occurrence of AMD is detected and / or determined by the extension of the primer with the DNA polymerase. In one embodiment, a polynucleotide primer of the invention hybridizes immediately adjacent to the variation. A single-base sequencing reaction using labeled dideodeoxynucleotide terminators can be used to detect variation. The presence of a variation will result in the inoration of the marked terminator, while the absence of a variation will not result in the inoration of the terminator. In another embodiment, a polynucleotide primer of the invention hybridizes to a variation in LOC387715, a variation in the SYNPR gene that is elated with AMD, or a variation in the PDGFC gene that is elated with the onset of AMD. The primer or a portion thereof will not hybridize to the LOC387715, SYNPR or PDGFC genes that do not contain the variation that is elated with the AMD. The presence of a variation will result in the extension of the primer, while the absence of a variation will not result in the extension of the primer. The primers and / or nucleotides can also include fluorescent, radioactive or chemical probes. A primer marked by the extension of the primer can be detected by measuring the intensity of the extension product, such as by gel electrophoresis, mass spectrometry or any other method to detect fluorescent, radioactive or chemical labels. In another embodiment, the presence of a variation in the LOC387715, SYNPR or PDGFC gene that is elated with the appearance of the AMD is detected and / or determined by ligation. In one embodiment, a polynucleotide primer of the invention hybridizes to a variation in the LOC387715, SYNPR or PDGFC gene that is elated with the onset of AMD. The primer, or a portion thereof, will not hybridize to a LOC387715, SYNPR or PDGFC gene that does not contain the variation. A second polynucleotide that hybridizes to a region of the LOC387715, SYNPR or PDGFC gene, immediately adjacent to the primer is also provided. One or both of the polynucleotide primers may be fluorescent, radioactive or chemical labeled. The ligation of the two polynucleotide primers will occur in the presence of DNA ligase if a variation in the LOC387715, SYNPR or PDGFC gene that is elated with the onset of AMD is present. The ligation can be detected by gel electrophoresis, mass spectrometry or by measuring the intensity of the fluorescent, radioactive or chemical labels. In another embodiment, the presence of a variation in the LOC387715, SYNPR or PDGFC gene that is elated with the occurrence of AMD is detected and / or determined by single-base extension (SBE). For example, a fluorescently labeled primer that is coupled with fluorescence resonance energy transfer (FRET) between the added base label and the primer label can be used. Typically, the method, such as that described by Chen et al., (PNAS 94: 10756-61 (1997), inorated herein by reference) utilizes a site-specific polynucleotide primer labeled at the 5'-terminus with 5-carboxyfluorescein (FAM) This labeled primer is designed so that the 3 'end is immediately adjacent to the polymorphic site of interest. The labeled primer hybridizes to the site, and a single base extension of the labeled primer is made with fluorescently labeled dideoxyribonucleotides (ddNTP) in a manner of sequencing the dye terminator, except that the deoxyribonucleotides are not present. An increase in the fluorescence of the aggregated ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity of the aggregated nucleotide. Methods to detect a variation in the LOC387715 gene, SYNPR or PDGFC that is correlated with the appearance of AMD may include amplification of a region of DNA comprising variation. Any method of amplification can be used. In a specific embodiment, a DNA region comprising the variation is amplified using the polymer chain reaction (PCR). PCR was initially described by Mullis (See, for example, U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, incorporated herein by reference), which discloses a method for increasing the concentration of a DNA region, in a mixture of genomic DNA, without cloning or purification. Other PCR methods can also be used for nucleic acid amplification, including but not limited to RT-PCR, quantitative PCR, real-time PCR, Rapid Amplified Polymorphic DNA Analysis, Rapid Amplification of the cDNA Ends (RACE) or amplification of encircling circle. For example, the polynucleotide primers of the invention are combined with a mixture of DNA (or any polynucleotide sequence that can be amplified with the polynucleotide primers of the invention), wherein the DNA comprises the LOC387715, SYNPR or PDGFC gene. The mixture also includes the necessary amplification reagents (eg, deoxyribonucleotide triphosphates, buffer, etc.), necessary for the thermal cycling reaction. In accordance with standard PCR methods, the mixture is subjected to a series of steps of denaturation, primer annealing and polymerase extension to amplify the DNA region comprising the variation in the LOC387715, SYNPR or PDGFC gene. The length of the amplified DNA region is determined by the relative positions of the primers one with respect to the other, and therefore, this length is a controllable parameter. For example, hybridization of the primers may occur, so that the ends of the primers, proximal to the variation, are separated by 1 to 10,000 base pairs (e.g., 10 base pairs (bp) 50 bp, 200 bp , 500 bp, 1, 000 bp, 2,500 bp, 5,000 bp or 10,000 bp). Standard instrumentation known to those skilled in the art is used for the amplification and detection of amplified DNA. For example, a wide variety of instrumentation has been developed to carry out nucleic acid amplifications, particularly PCR, for example, Johnson et al, U.S. Pat. No. 5,038,852 (computer controlled thermal cycler); Wittwer et al, Nuclear Acids Research, 17: 4353-4357 (1989) (PCR with capillary tube); Hallsby, Patent of E.U.A. No. 5,187,084 (temperature control based on air); Garner et al, Biotechniques, 14: 112-115 (1993) (High performance PCR in 864-well plates); Wildíng et al, International Application No. PCT / US93 / 04039 (PCR in micromachined structures); Schnipelsky et al, European Patent Application No. 90301061.9 (publication No. 0381501 A2) (disposable, single-use PCR device). In certain embodiments, the invention described herein utilizes real-time PCR or other methods known in the art, such as the Taqman assay. In certain embodiments, a LOC387715, SYNPR or variant PDGFC gene, which is correlated with the occurrence of AMD in humans can be detected using polymorphism analysis of the single-stranded configuration, which identifies differences in bases by altering the electrophoretic migration of the single-stranded PCR products, as described in Oríta et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single-strand amplification products. The single-stranded nucleic acids can refold or form secondary structures that are partially dependent on the sequence of the base. The different electrophoretic mobilities of the single-strand amplification products can be related to the differences in base sequence between the alleles of the target sequences.

In one embodiment, the amplified DNA is analyzed in conjunction with one of the detection methods described herein, such as by DNA sequence sequencing. The amplified DNA can be analyzed alternatively by hybridization with a labeled probe, hybridization to an array or DNA microarray, by the incorporation of the biotinylated primers, followed by the detection of the avidin-enzyme conjugate, or by incorporation of the 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, in the amplified segment. In a specific embodiment, the amplified DNA is analyzed by determining the length of the amplified DNA by electrophoresis or chromatography. For example, the amplified DNA is analyzed by gel electrophoresis. Gel electrophoresis methods are well known in the art. See, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992. The amplified DNA can be visualized, for example, by fluorescent or radioactive means, or with other dyes or markers that intersperse the DNA. The DNA can also be transferred to a solid support such as a nitrocellulose membrane and subjected to Southern blotting after gel electrophoresis. In one embodiment, the DNA is exposed to ethidium bromide and visualized under ultraviolet light.

Therapeutic nucleic acids encoding the LOC387715, SYNPR and PDGFC polypeptides In certain embodiments, the invention provides isolated and / or recombinant nucleic acids encoding a LOC387715 polypeptide, a SYNPR polypeptide or a PDGFC polypeptide, including the functional variants, described herein. The subject nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. These nucleic acids can be used, for example, in methods for making the LOC387715, SYNPR or PDGFC polypeptides, or as direct therapeutics (for example, in a gene therapy approach). It is further understood that the subject nucleic acids encoding the LOC387715, SYNPR or PDGFC polypeptides include nucleic acids that are variants of the sequences available to the public (eg, through databases) and the sequences referred to herein. Variant nucleotide sequences that differ by one or more substitutions, additions or nucleotide deletions, such as allelic variants, and will therefore include the coding sequences that differ from the nucleotide sequence of the coding sequence or the coding sequences available to the public, referred to herein. In certain embodiments, the invention provides isolated or recombinant nucleic acid sequences that are complementary to, or at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequences of nucleic acids available to the public or to the nucleic acid sequences referred to herein. In the further embodiments, the nucleic acid sequences of the invention can be isolated, recombinant and / or fused to a heterologous nucleotide sequence or in a DNA library. In other embodiments, the nucleic acids of the invention also include nucleic acids that hybridize under stringent conditions to the nucleotide sequences available to the public or to the nucleic acid sequences referred to herein or fragments thereof. As discussed above, one of ordinary skill in the art will readily understand that the appropriate stringent conditions that promote DNA hybridization may vary. For example, one could hybridize at 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a 2.0 x wash of SSC at 50 ° C. For example, the concentration of the salt in the wash step can be selected from low stringency of about 2.0 x SSC at 50 ° C at high stringency of about 0.2 x SSC at 50 ° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 ° C, to high stringency conditions at about 65 ° C. Both the temperature and the salt can vary, either the temperature or the concentration of the salt can be kept constant, while the other variable is changed. In one embodiment, the invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature, followed by a 2 x wash of SSC at room temperature. Isolated nucleic acids that differ from the nucleic acids described herein due to degeneracy in the genetic code are also within the scope of the invention. For example, several amino acids are designated by more than one triplet. Codons that specify the same amino acid or synonyms (for example, CAU and CAC are synonyms for histidine), can result in "silent" variations that do not affect the amino acid sequence of the protein. However, it is expected that polymorphisms of the DNA sequence that do not lead to changes in the amino acid sequences of the subject proteins, exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among the individuals of a given species due to allelic variation. natural. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. The nucleic acids and polypeptides of the invention can be produced using standard recombinant methods. For example, the recombinant nucleic acids of the invention can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional initiation and termination sequences, translation initiation and termination sequences and enhancer or activating sequences. Constitutive or inducible promoters as are known in the art are contemplated by the invention. The promoters can be natural promoters or hybrid promoters that combine the elements of more than one promoter. An expression construct may be present in a cell in an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. The expression vector may also contain a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. In certain embodiments of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a LOC387715 polypeptide, a SYNPR polypeptide, or a PDGFC polypeptide and which is operably linked to at least one regulatory sequence . Regulatory sequences are recognized in the art, and are selected to direct the expression of LOC387715, SYNPR or PDGFC polypeptide. The term "regulatory sequence" includes promoter, enhancer, terminator sequences, preferred sequences of the ribosome binding site, preferred mRNA leader sequences, preferred protein processing sequences, preferred signal sequences for secretion of the protein and other elements of expression control. Examples of regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For example, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operably linked thereto can be used in these vectors to express the DNA sequences encoding a LOC387715 polypeptide. , SYNPR or PDGFC. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the immediate initial promoter of adenovirus or cytomegalovirus, RSV promoters, the lac system, the trp system, the TAC or TRC system. , the T7 promoter whose expression is directed by T7 RNA polymerase, the major regions of the operator and the lambda phage promoter, the control regions for the fd coating protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the acid phosphatase, for example, Pho5, the promoters of the factors that coincide with yeast, the polyhedral promoter of the baculovirus system and other sequences known to control the expression of the genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the number of copies of the vector, the ability to control that number of copies and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. A recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammal), or both. Expression vehicles for the production of recombinant LOC387715, SYNPR or PDGFC polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the types: plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from pEX, plasmids derived from pBTac and plasmids derived from pUC for expression in prokaryotic cells, such as E. coli. Some mammalian expression vectors contain prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences of bacterial plasmids, such as pBR322, to facilitate replication and selection of drug resistance in prokaryotic and eukaryotic cells. Alternatively, virus derivatives such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, derived from pREP and p205), can be used for the transient expression of proteins in cells eukaryotic Examples of other viral expression systems (including retroviral) can be found below in the description of the gene therapy delivery systems. The various methods employed in the preparation of plasmids and in the transformation of host organisms are well known in the art. For other expression systems suitable for prokaryotic and eukaryotic cells, as well as for general recombinant methods, see Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001). In some cases, it may be desirable to express the recombinant polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393 and pVL941), vectors derived from pAcUW (such as pAcUWI), and vectors derived from pBlueBac (such as pBlueBac III containing β-gal). In one embodiment, a vector will be designed for the production of a polypeptide (eg, a LOC387715, SYNPR or PDGFC polypeptide) in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), PcDNA4 vectors (Invitrogen, Carlsbad, Calif.) And the pCI-neo vectors (Promega, Madison, Wise). In other embodiments, the vector is designed for the production of a polypeptide (e.g., a LOC387715, SYNPR or PDGFC polypeptide) in procatic host cells (e.g., E. coli and B. subtilis), eukaryotic host cells such as, for example, yeast cells, insect cells, myeloma cells, 3T3 fibroblast cells, monkey or COS kidney cells, mink lung epithelial cells, human scrotum fibroblast cells, human glioblastoma cells, and teratocarcinoma Alternatively, the genes can be expressed in a cell-free system, such as the rabbit reticulocyte lysate system. The subject gene constructs can be used to express the LOC387715, SYNPR or PDGFC polypeptides in cells propagated in culture, for example, to produce proteins, including fusion proteins or variant proteins, for purification. This invention also pertains to a host cell transfected with a recombinant gene that includes a coding sequence for the LOC387715, SYNPR or PDGFC polypeptides. The host cell can be any prokaryotic or eukaryotic cell. For example, a LOC387715, SYNPR or PDGFC polypeptide of the invention can be expressed in bacterial cells, such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells. Other suitable host cells are known to those skilled in the art. The present invention also pertains to methods for producing the LOC387715, SYNPR or PDGFC polypeptides. For example, a host cell transfected with an expression vector encoding a LOC387715, SYNPR or PDGFC polypeptide, can be cultured under appropriate conditions to allow expression of the LOC387715, SYNPR or PDGFC polypeptide to occur. The LOC387715, SYNPR or PDGFC polypeptides can be secreted and isolated from a mixture of cells and a medium containing the LOC387715, SYNPR or PDGFC polypeptides. Alternatively, the polypeptide can be retained cytoplasmically or in a membrane fraction, the cells are harvested and lysed and the protein isolated. A cell culture includes host cells, medium and other by-products. The suitable medium for cell culture is known in the art. The polypeptide can be isolated from a cell culture medium, host cells or both, using techniques known in the art to purify proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and immunoaffinity purification with specific antibodies. for the particular epitopes of the polypeptide. In a particular embodiment, the polypeptide LOC387715, SYNPR or PDGFC is a fusion protein containing a domain that facilitates the purification of LOC387715, SYNPR or PDGFC polypeptide.

In another embodiment, a fusion gene encoding a leader purification sequence, such as the poly- (His) sequence / enterokinase cleavage site at the N-terminus of the desired portion of the LOC387715 polypeptide, SYNPR or recombinant PDGFC, can allow the purification of the expressed fusion protein by affinity chromatography using a Ni2 + metal resin. The leader purification sequence can be subsequently removed by treatment with enterokinase to provide the purified polypeptide (for example, see Hochuli et al., (1987) J. Chromatography 41 1: 177; and Janknecht et al., PNAS USA 88: 8972 ). The techniques for making the fusion genes are well known.

Essentially, the binding of several DNA fragments encoding the different polypeptide sequences is performed according to conventional techniques, using terms with blunt ends or stepped ends for ligation, digestion with the restriction enzyme to provide the appropriate terms, filling the Cohesive ends as appropriate, treatment with alkaline phosphatase to avoid undesirable binding and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automatic DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be carried out using anchoring primers that give rise to complementary overhangs between two consecutive gene fragments that can be subsequently annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds, Ausubel et al., John Wiley &Sons: 1992).

Antisense Polynucleotides In certain embodiments, the invention provides polynucleotides comprising an antisense sequence that acts through an antisense mechanism to inhibit the expression of a variant LOC387715, SYNPR or PDGFC gene. Antisense technologies have been widely used to regulate gene expression (Buskírk et al., Chem Biol 11, 1157-63 (2004); and Weiss et al., Cell Mol Life Sci 55, 334-58 (1999)). As used herein, "antisense" technology refers to the administration or in situ generation of molecules or their derivatives that hybridize specifically (e.g., bind) under cellular conditions, with the target nucleic acid of interest (mRNA and / or genomic DNA), which encodes one or more of the target proteins to inhibit the expression of that protein, for example, inhibiting transcription and / or translation, such as by spherical hindrance, altering splicing or induction of the excision or other enzymatic inactivation of the transcript. The binding can be by conventional complementarity of the base pairs, or, for example, in the case of DNA duplex binding, through specific interactions in the main cleavage of the double helix. A polynucleotide comprising an antisense sequence of the present invention can be delivered, for example, as a component of an expression plasmid which, when transcribed in the cell, produces a nucleic acid sequence that is complementary to at least a single portion of the target nucleic acid. Alternatively, the polynucleotide comprising an antisense sequence can be generated outside the target cell, and which, when introduced into the target cell, causes inhibition of expression by hybridizing to the target nucleic acid. The polynucleotides of the invention can be modified such that they are resistant to endogenous nucleases, for example, exonucleases and / or endonucleases, and therefore, are stable in vivo. Examples of nucleic acid molecules for use in the polynucleotides of the invention are DNA phosphoramidate, phosphothioate and methylfosphonate analogs (see also U.S. Patent Nos. 5, 176,996, 5,264,564, and 5,256,775). General approaches for constructing polynucleotides useful in antisense technology have been reviewed, for example, by van der Krol et al. (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res 48: 2659-2668. Antisense approaches involve the design of polynucleotides (either DNA or RNA) that are complementary to a target nucleic acid encoding a LOC387715, SYNPR or variant PDGFC gene. The antisense polynucleotide can bind to an mRNA transcript and prevent translation of a protein of interest. Absolute complementarity, although preferred, is not required. In the case of double-stranded antisense polynucleotides, a single strand of the duplex DNA can be tested as such, or the triple formation can be tested. The ability to hybridize will depend on the degree of complementarity and the length of the antisense sequence. Generally, the longer the hybridizing nucleic acid is, the more mismatches with the base with a target nucleic acid may contain, and still form a stable (or triple, as the case may be) duplex. One of skill in the art can determine a tolerable degree of mismatches by using standard procedures to determine the melting point of the hybridized complex. Antisense polynucleotides that are complementary to the 5 'end of a target mRNA, for example, the 5' untranslated sequence - until a, and including the AUG start codon, must work more poorly to inhibit mRNA translation. However, the sequences complementary to the 3 'untranslated sequences of the mRNAs have recently been shown to be effective in inhibiting the translation of the mRNAs as well (Wagner, R. 1994. Nature 372: 333). Therefore, antisense polynucleotides complementary to the 5 'or 3' non-translated non-coding regions of a LOC387715, SYNPR or variant PDGFC gene can be used in an antisense approach to inhibit translation of a variant LOC387715, SYNPR or PDGFC mRNA. Antisense polynucleotides complementary to the 5 'untranslated region of an mRNA must include the complement of the AUG start codon. Antisense polynucleotides complementary to the mRNA coding regions are less efficient inhibitors of translation, but may also be used according to the invention. Whether designed to hybridize to the 5 ', 3' coding region of the mRNA, the antisense polynucleotides must be at least six nucleotides in length, and are preferably less than about 100 and more preferably less than about 50. , 25, 17 or 10 nucleotides in length. Regardless of the choice of the target sequence, it is preferred that the in vitro studies are performed first to quantify the ability of the antisense polynucleotide to inhibit the expression of a variant LOC387715, SYNPR or PDGFC gene. It is preferred that these studies utilize the controls that distinguish between inhibition of the antisense gene and non-specific biological effects of the antisense polynucleotide. It is also preferred that these studies compare the levels of the target RNA or protein with those of an RNA or internal control protein. Furthermore, it is considered that the results obtained using the antisense polynucleotide are compared with those obtained using a control antisense polynucleotide. It is preferred that the control antisense polynucleotide be about the same length as the test antisense polynucleotide and that the nucleotide sequence of the antisense control polynucleotide differ from the antisense sequence of interest no more than necessary to avoid sequence-specific hybridization. objective. The polynucleotides of the invention, including the antisense polynucleotides, can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The polynucleotides of the invention can be modified in the portion of the base, the sugar portion or the phosphate backbone, for example, to improve the stability of the molecule, hybridization, etc. The polynucleotides of the invention can include other adjoining groups such as peptides (for example, to select host cell receptors), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. , 1989, Proc Nati Acad Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc Nati Acad Sci. USA 84: 648-652; PCT Publication No. W088 / 09810, published on December 15, 1988 ) or the blood-brain barrier (see, for example, PCT Publication No. W089 / 10134, published April 25, 1988), cleavage agents activated by hybridization. (See, for example, Krol et al., 1988, BioTechniques 6: 958-976) or intercalating agents. (See, for example, Zon, Pharm, Res. 5: 539-549 (1988)). For this purpose, a polynucleotide of the invention can be conjugated to another molecule, for example, a peptide, a cross-linking agent activated by hybridization, a transport agent, a cleavage agent activated by hybridization, etc. The polynucleotides of the invention, including the antisense polynucleotides, may comprise at least a portion of the modified base selected from the group including non-exclusively 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxytriethyl) uracil, -carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-? Sopenten? Laden? Na, 1-met? Lguan? A, 1-met? L? Nosena, 2 , 2-d? Met? Lguan? Na, 2-met? Laden? Na, 2-met? Lguan? Na, 3-met? Lc? Tos? Na, 5-met? Lc? Tos? Na, N6-aden na, 7-met? lguan? na, 5-met? lam? nom? lurac? lo, 5-methox? am? nom? l-2-t? ourac? lo, beta-D-mannosilqueosine, 5-methox carboxymethyl methacrylate, 5-methoxuracury, 2-methytho-N6-? arepenten? laden? na, urac? l-5-ox? acetic acid (v), wibutoxosine, pseudouracil, queosine, 2-t? oc? tos? na, 5-met? l-2-t? ourac? lo, 2-t? ourac? lo, 4-t? ourac? lo, 5-met? lurac? te, methyl ester of urac? l-5-ox? acetic acid, urac? l-5-ox? acetic acid (v), 5-met? l-2-t? ourac? lo, 3- (3-am? No-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-d? Am? Nopur? Na The polynucleotides of the invention may also comprise at least a portion of modified sugar selected from the group that includes non-exclusively arabinose, 2-fluoroarabose, xylulose and hexose U The polynucleotide of the invention may also contain a neutral peptide-like backbone such molecules are referred to as peptide nucleic acids (PNA) -ol? gomers and are described, for example, in Perry-O'Keefe et al (1996) Proc Nati Acad Sci USA 93 14670 and in Eglom et al (1993) Nature 365 566 An advantage of PNA oligomers is their ability to bind complementary DNA in an essentially independent manner from the ionic strength of the medium due to the neutral DNA backbone. Another embodiment, a nucleotide po of the invention comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphoniamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal. or analogous thereof. In a further embodiment, the polynucleotides of the invention, including the antisense polynucleotides, are anomeric oligonucleotides. An anomeric oligonucleotide forms specific double-stranded hybrids with the complementary RNA, in which, contrary to the usual units, the strands run parallel to one another (Gautier et al., 1987, Nucí Acids Res. : 6625-6641). The oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., 1987, Nucí Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. : 327-330). The polynucleotides of the invention, including antisense polynucleotides, can be synthesized by standard methods known in the art, for example, by the use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the methods of Stein et al. Nucí Acids Res. 16: 3209 (1988)), methylphosphonate oligonucleotides can be prepared by the use of controlled pore glass polymer supports (Sarin et al., Proc. Nati, Acad. Sci. USA 85: 7448-7451 (1988 )), etc. Antisense sequences complementary to the coding region of an mRNA sequence can be used. Alternatively, those complementary to the transcribed untranslated region and to the region comprising the starting methionine can be used. Antisense polynucleotides can be delivered to the cells expressing the target m genes. Several methods have been developed to deliver the nucleic acids to the cells, for example, can be injected directly into the tissue site, or modified nucleic acids, designed to target the desired cells (eg, antisense polynucleotides linked to peptides or antibodies that specifically bind to the expressed receptors or antigens) on the surface of the target cell), can be administered systematically However, it can be difficult to reach the intracellular concentrations of the antisense nucleotides, sufficient to attenuate the activity of a variant LOC387715, SYNPR or PDGFC gene or mRNA in certain cases. Therefore, another method uses a recombinant DNA construct in which the antisense polynucleotide is placed under the control of a poly III or poly promoter. Strong II The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of antisense polynucleotides that will form base pairs complementary to the variant LOC387715, SYNPR or PDGFC gene or mRNA, and therefore attenuate the activity of the protein LOC387715, SYNPR or PDGFC For example, a vector can be introduced in vivo so that it is picked up by a cell and directs the transcription of an antisense polynucleotide that selects a gene or mRNA LOC387715, SYNPR or variant PDGFC. Such a vector can remain episomal or be chromosomally integrated, as long as it can be transcribed to produce the desired antisense polynucleotide. Such vectors can be constructed by standard recombinant DNA technology methods in the art. The vectors can be a plasmid, viral or others known in the art, used for replication and expression in mammalian cells. A promoter can be operably linked to the sequence encoding the antisense polynucleotide. The expression of the sequence encoding the antisense polynucleotide can be any promoter known in the art to act on mammalian, preferably human, cells. Such promoters may be inducible or constitutive. Such promoters include, but are not limited to: the SV40 initial promoter region (Bernoist and Chambon, Nature 290: 304-310 (1981)), the promoter contained in the 3 'long terminal repeat of the Rous sarcoma virus ( Yamamoto et al, Cell 22: 787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Nati, Acad. Sci. USA 78: 1441-1445 (1981)), the sequences regulators of the metallothionin gene (Brinster et al, Nature 296: 3942 (1982)), etc. Any type of plasmid, cosmid, YAC or viral vector to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors selectively infecting the desired tissue can be used, in which case administration can be achieved by another route (eg, systemically).

RNAi-siRNA and mRNA constructs RNA interference (RNAi) is a phenomenon that describes the specific post-transcriptional silencing of the double-stranded RNA-dependent gene (ds). The present invention provides a polynucleotide comprising an RNAi sequence that acts through an RNAi or mRNA mechanism to attenuate the expression of a variant LOC387715, SYNPR or PDGFC gene. For example, a polynucleotide of the invention may comprise an mRNA or siRNA sequence that attenuates or inhibits the expression of a variant LOC387715, SYNPR or PDGFC gene. In one embodiment, the mRNA or siRNA sequence is between about 19 nucleotides and about 75 nucleotides in length, or preferably, between about 25 base pairs and about 35 base pairs in length. In certain embodiments, the polynucleotide is a hairpin spiral or stem spiral that can be processed by RNase enzymes (e.g., Drosha and Dicer). An RNAi construct contains a nucleotide sequence that hybridizes under physiological conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for a variant LOC387715, SYNPR or PDGFC gene. The double-stranded RNA only needs to be sufficiently similar to the natural RNA that has the ability to mediate RNAi. The number of poor tolerated mappings of the nucleotide between the target sequence and the sequence of the RNAi construct is no more than 1 in 5 base pairs, or 1 in 10 base pairs, or 1 in 20 base pairs, or 1 in 50 base pairs It is important in a primary way that the RNAi construct is able to select specifically a LOC387715, SYNPR or vanishing PDGFC gene. Mismatches in the center of the siRNA duplex are more critical and can essentially cancel RNA cleavage In contrast, the nucleotides at the 3 'end of the RNAi strand that is complementary to the target RNA do not contribute significantly to the specificity of the target recognition. The identity of the sequence can be optimized by comparing the sequence and the alignment algorithms known in the art (see Gnbskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein), and ca Calculate the percent difference between nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT program using the default parameters (eg, Genetic Computation Group of the University of Wisconsin). of 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the target gene portion. Alternatively, the duplex region of the RNA can be functionally defined as a nucleotide sequence that is capable of hybridize with a portion of the target gene transcript (eg, 400 mM NaCI, 40 mM PIPES, pH 6 4, 1 mM EDTA, hybridization at 50 ° C or 70 ° C for 12-16 hours, followed by washing). of the nucleotides comprising the RNAi sequences can be carried out by a variety of methods. For example, polynucleotides comprising the RNAi sequences can be produced by synthetic chemical methods or by recombinant nucleic acid techniques. The endogenous polymeric RNA of the treated cell can mediate transcription in vivo, or the cloned RNA polymerase can be used for in vitro transcription. Polynucleotides of the invention, including wild-type or antisense polynucleotides, or those that modulate the activity of the target gene by RNAi mechanisms, can include modifications to the phosphate-sugar or nucleoside backbone, for example, to reduce the susceptibility to cellular nucleases, improve bioavailability, improve the characteristics of the formulation and / or change other pharmacokinetic properties. For example, the phosphodiester bonds of the natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. Modifications in RNA structure can be designed to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, the bases can be modified to block the activity of adenosine deaminase. The polynucleotides of the invention can be produced enzymatically or by partial / total organic synthesis, any modified ribonucleotide can be introduced by enzymatic or organic synthesis in vitro. Methods for chemically modifying RNA molecules can be adapted to modify the RNAi constructs (see, for example, Heidenreich et al (1997) Nucleic Acids Res, 25: 776-780; Wilson et al. (1994) J Mol Recog 7: 89-98; Chen et al. (1995) Nucleic Acids Res 23: 2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7: 55-61). Merely to illustrate, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate phosphodiesters, peptide nucleic acids, oligomers containing 5-propynyl-pyrimidine or modifications in sugar (eg, substituted ribonucleosides) in position 2 ', configuration a). The double-stranded structure can be formed by a single strand of self-complementary RNA or two self-complementary RNA strands. The RNA duplex formation can start either inside or outside the cell. The RNA can be introduced in an amount that allows the delivery of at least one copy per cell. Higher doses (eg, at least 5, 10, 100, 500 or 1000 copies per cell) of the double-stranded material may provide more effective inhibition, while lower doses may also be useful for specific applications. The inhibition is specific to the sequence in which the nucleotide sequences corresponding to the duplex region of the RNA are selected for genetic inhibition. In certain embodiments, the object RNAi constructs are "ARNsi". These nucleic acids are between about 19-35 nucleotides in length, and even more preferably, 21-23 nucleotides in length, for example, which correspond in length to the fragments generated by the "fragmentation" of the nuclease of the Longest double-stranded RNA. It is understood that siRNAs recruit the nuclease complexes and guide the complexes to the target mRNA by mating to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex or the translation is inhibited. In a particular embodiment, siRNA molecules of 21-23 nucleotides comprise a 3 'hydroxy group. In other embodiments, the object RNAi constructs are "RNAi". MicroRNAs (RNAi) are small non-coding RNAs that direct the transcriptional regulation of gene expression through interaction with homologous mRNAs. The miRNA controls the expression of genes by binding to the complementary sites in the target mRNAs of the genes encoding the protein. The mRNAs are similar to the siRNAs. The miRNAs are processed by the nucleolithic cleavage of longer double-stranded precursor molecules. These precursor molecules are often hairpin structures of about 70 nucleotides in length, with 25 or more nucleotides that are paired to the base in the hairpin. Enzymes similar to RNase III, Drosha and Dicer (which can also be used in siRNA processing) cleave the miRNA precursor to produce an RNAi. The processed mRNA is single-stranded and incorporated into a protein complex, termed RISC or miRNP. This RNA-protein complex selects a complementary mRNA. The mRNAs inhibit the translation or direct excision of the target mRNAs. (Brennecke et al., Genome Biology 4: 228 (2003); Kim et al., Mol.Cells 19: 1-15 (2005).) In certain embodiments, the RNAi and siRNA constructs can be generated by RNA processing. longer double strands, for example, in the presence of Dicer or Drosha enzymes Dicer and Drosha are RNase III-like nucleases that specifically cleave dsRNA Dicer has a distinctive structure that includes a helicase domain and double motifs of RNAse III Dicer also contains a region of homology for the family RDE1 / QDE2 / ARGONAUTE, which has been genetically related to AENi in lower eukaryotes.In reality, the activation of, or overexpression of Dicer may be sufficient in many cases to allow RNA interference in otherwise non-receptive cells, such as cultured eukaryotic cells or mammalian (non-oocytic) cells in culture or in whole organisms.The methods and compositions they employ Dicer, as well as other RNAi enzymes, are described in the Patent Application Publication of E.U.A. No. 2004/0086884. In one embodiment, the in vitro Drosophila system is used. In this embodiment, a polynucleotide comprising an RNAi sequence or an RNAi precursor is combined with a soluble extract derived from Drosophila embryos, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides. RNAmi and siRNA molecules can be purified using various techniques known to those skilled in the art. For example, gel electrophoresis can be used to purify such molecules. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA and mRNA molecules. In addition, chromatography (for example, size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody, can be used to purify the siRNAs and the miRNAs. In certain embodiments, at least one strand of the siRNA sequence of an effector domain has a 3 'overhang of about 1 to about 6 nucleotides in length, or 2 to 4 nucleotides in length. In other embodiments, the 3 'overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a projection 3 'and the other strand is blunt end or also has a projection. The length of the protrusions can be the same or different for each strand. In order to further improve the stability of the siRNA sequence, the 3 'overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of the pyrimidine nucleotides with the modified analogs, for example, substitution of the 3 'overhangs of the uridine nucleotide with 2'-deoxythymidine, is tolerated, and does not affect the efficiency of the RNAi. The absence of a 2 'hydroxyl significantly improves the nuclease resistance of the overhang in the tissue culture medium and may be beneficial in vivo. In certain embodiments, a polynucleotide of the invention comprising an RNAi sequence or an RNAi precursor is in the form of a hairpin structure (referred to as a hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from the RNA polymerase III promoters in vivo. Examples for making and using such hairpin RNAs for silencing a gene in mammalian cells are described, for example, Paddison et al, Genes Dev, 2002, 16: 948-58; McCaffrey et al., Nature, 2002, 418: 38-9; McManus et al., RNA 2002, 8: 842-50; Yu et al., Proc Nati Acad Sci USA, 2002, 99: 6047-52). Preferably, such hairpin RNAs are designed in cells or in an animal to ensure the continuous and stable suppression of a desired gene. It is known in the art that mRNA and siRNA can be produced by processing a hairpin RNA in the cell. In still other embodiments, a plasmid is used to deliver the double-stranded RNA, for example, as a transcriptional product. After the coding sequence is transcribed, the complementary RNA transcripts are paired with the bases to form the double-stranded RNA.

Antibodies Another aspect of the invention pertains to antibodies. In one embodiment, an antibody that is specifically reactive with a LOC387715, SYNPR or variant PDGFC polypeptide can be used to detect the presence of a variant LOC387715, SYNPR or PDGFC polypeptide or to inhibit the activity of a variant LOC387715, SYNPR or PDGFC polypeptide. . For example, by using immunogens derived from a LOC387715, SYNPR or variant PDGFC peptide, an antiprotein / antisense antiserum or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or a rabbit can be immunized with an immunogenic form of the LOC387715 peptide, variant SYNPR or PDGFC, an antigenic fragment that is capable of eliciting an antibody or fusion protein response. In a particular embodiment, the inoculated mouse does not express LOC387715, SYNPR or endogenous PGDFC, thus facilitating the isolation of antibodies that would otherwise be eliminated as anti-self antibodies. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of a LOC387715, SYNPR or variant PDGFC peptide can be administered in the presence of an adjuvant. The progress of the immunization can be verified by the detection of antibody titers in the plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as the antigen to assess antibody levels. After immunization of an animal with an antigenic preparation of a LOC387715, SYNPR or variant PDGFC polypeptide, the antiserum can be obtained, and if desired, the polyclonal antibodies can be isolated from the serum. To produce the monoclonal antibodies, the cells that produce antibodies (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion methods with immortalized cells such as myeloma cells to provide the hybridoma cells. Such techniques are well known in the art, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the hybridoma technique of human B lymphocytes (Kozbar et al. , (1983) Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Colé et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77- 96). Hybridoma cells can be selected immunochemically for the production of antibodies reactive specifically with a LOC387715, SYNPR or variant PDGFC polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells. The term "antibody" as used herein is intended to include fragments thereof that are also specifically reactive with a variant LOC387715, SYNPR or PDGFC polypeptide. Antibodies can be fragmented using conventional techniques and fragments selected for utility, in the same manner as described above for whole antibodies. For example, F (ab) 2 fragments can be generated by treating the antibody with pepsin. The resulting F (ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibodies of the present invention further aim to include bispecific, single-chain, and chimeric and humanized molecules that have affinity for a variant LOC387715, SYNPR or PDGFC polypeptide, conferred by at least one CDR region of the antibody. In preferred embodiments, the antibody further comprises a label attached thereto and capable of being detected (e.g., the label may be a radioisotope, a fluorescent compoundenzyme or enzyme cofactor). In certain embodiments, an antibody of the invention is a monoclonal antibody, and in certain embodiments, the invention makes available methods for generating new antibodies that specifically bind to the variant LOC387715, SYNPR or PDGFC polypeptides. For example, a method for generating a monoclonal antibody that specifically binds a LOC387715, SYNPR or variant PDGFC polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the LOC387715, SYNPR or PDGFC polypeptide, effective to stimulate a detectable immune response, obtain cells that produce antibodies (eg, spleen cells) from the mouse and fuse the cells that produce the antibody with myeloma cells to obtain hybridomas that produce an antibody, and test the hybridomas that produce an antibody to identify a hybridoma that produces a monoclonal antibody that binds specifically to the variant LOC387715, SYNPR or PDGFC polypeptide. Once obtained, the hybridoma can be propagated in a cell culture, optionally under culture conditions where cells derived from the hybridoma produce the monoclonal antibody that binds specifically to the polypeptide LOC387715, SYNPR or PDGFC. The monoclonal antibody can be purified from the cell culture. The term "reactive specifically with", as used with reference to an antibody, is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (eg, a polypeptide LOC387715, SYNPR or PDGFC variant) and other antigens that are not of interest, for which the antibody is useful, detect the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of binding specificity may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and the cross-reactive polypeptides. A characteristic that influences the specificity of an antibody: antigen interaction is the affinity of the antibody for the antigen. Although the desired specificity can be achieved with a range of different affinities, the preferred antibodies will generally have an affinity (a dissociation constant) of about 10"6, 10" 7, 10 ~ 8, 10'9 or less.

Selection assays The present invention relates to the use of the LOC387715, SYNPR or PDGFC polypeptides to identify compounds (agents) that are agonists or antagonists of the LOC387715, SYNPR or PDGFC polypeptides. The compounds identified through this selection can be tested in the cells of the eye (e.g., epithelial and endothelial cells), as well as in other tissues (e.g., muscle and / or neurons) to assess their ability to modulate LOC387715 activity. , SYNPR or PDGFC in vivo or in vitro. In certain aspects, the compounds identified through this selection modulate the formation of druse deposits. Optionally, these compounds can be further tested in animal models to assess their ability to modulate the activity of LOC387715, SYNPR or PDGFC in vivo. There are numerous approaches for selecting therapeutic agents that select the LOC387715, SYNPR or PDGFC polypeptides. In certain embodiments, a high throughput screening of the compounds can be carried out to identify agents that affect the activity of the LOC387715, SYNPR or PDGFC polypeptides. A variety of test formats will suffice, and in light of the present disclosure, those not expressly described herein, however, will be understood by one skilled in the art. As described herein, the test compounds (agents) of the invention can be created by a combinatorial chemical method. Alternatively, the subject compounds can be natural biomolecules synthesized in vivo or in vitro. The compounds (agents) to be tested for their ability to act as modulators of the activity of LOC387715, SYNPR or PDGFC, can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), produced from chemical way (eg, small molecules, including peptidomimetics), or to be produced recombinantly. The test compounds contemplated by the present invention include organic molecules that are not peptidyl, peptides, polypeptides, peptidomimetics, sugars, hormones and nucleic acid molecules. The test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as those made by combinatorial chemistry. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other kinds of organic compounds. The presentation of the test compounds for testing the system may be in isolation or as mixtures of compounds, especially in the initial selection steps.

Optionally, the compounds can optionally be derivatized with other compounds and have derivative groups that facilitate isolation of the compounds. Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxigenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatable crosslinkers or any combination thereof.

Pharmaceutical compositions The methods and compositions described herein for treating a subject suffering from AMD can be used for the prophylactic treatment of individuals who have been diagnosed or predicted to be at risk of developing AMD. In this case, the composition is administered in an amount and dose that is sufficient to delay, slow down or prevent the onset of AMD or related symptoms. Alternatively, the methods and compositions described herein may be used for the therapeutic treatment of individuals suffering from AMD. In this case, the composition is administered in an amount and dose that is sufficient to delay or slow down the progression of the condition, in whole or in part, or in an amount and dose that is sufficient to reverse the condition to the point of eliminate the disorder. It is understood that an effective amount of a composition for treating a subject who has been diagnosed or predicted to be at risk of developing AMD, is a dose or amount that is in sufficient amounts to treat a subject or to treat the disorder itself. In certain embodiments, the compounds of the present invention (e.g., an isolated or recombinantly produced nucleic acid molecule encoding a LOC387715, SYNPR or PDGFC polypeptide or a LOC387715, SYNPR or PDGFC polypeptide isolated or recombinantly produced), they are formulated with a pharmaceutically acceptable carrier. For example, a LOC387715, SYNPR or PDGFC polypeptide or a nucleic acid molecule encoding a LOC387715, SYNPR or PDGFC polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition). The subject compounds can be formulated for administration in any convenient way for use in human medicine. In certain embodiments, the therapeutic methods of the invention include administering the composition topically, systemically or locally. For example, the therapeutic compositions of the invention may be formulated for administration by, for example, injection (eg, intravenously, subcutaneously or intramuscularly), inhalation or insufflation (either through the mouth or nose) or administration oral, buccal, sublingual, transdermal, nasal or parenteral. The compositions described herein may be formulated as part of an implant or device. When administered, the therapeutic composition for use in this invention is in a physiologically acceptable pyrogen-free form. In addition, the composition can be encapsulated or injected in a viscous form for delivery to the site where the target cells are present, such as to eye cells. The techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. In addition to the LOC387715, SYNPR or PDGFC polypeptides or the nucleic acid molecules encoding the LOC387715, SYNPR or PDGFC polypeptides, therapeutically useful agents may optionally be included in any of the compositions as described above. In addition, therapeutically useful agents can, alternatively or additionally, be administered simultaneously or sequentially with the polypeptides LOC387715, SYNPR or PDGFC or the nucleic acid molecules encoding the LOC387715, SYNPR or PDGFC polypeptides, according to the methods of the invention. In certain embodiments, the compositions of the invention may be administered orally, for example, in the form of capsules, seals, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules or as a solution or a suspension in an aqueous or non-aqueous liquid, or in an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as a tablet (using an inert base, such as gelatin and glycerin or sucrose and acacia) and / or as mouthwashes and the like, each containing a predetermined amount of an agent as an active ingredient. An agent can also be administered as a bolus, confection or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more therapeutic compounds of the present invention can be mixed with one or more pharmaceutically acceptable carriers, such as citrate sodium or dicalcium phosphate and / or any of the following (1) fillers or entenders, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid, (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia, (3) humectants, such as glycerol, (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and carbonate sodium, (5) agents that retard the solution, such as paraffin, (6) absorption accelerators, such as quaternary ammonium compounds, (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, (8) sorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof and (10) ) Coloring Agents In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be used as fillings in filled soft and hard gelatin capsules, using excipients such as lactose or sugars. of milk, as well as high molecular weight polyethylene glycols and the like Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol , benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to the inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. The suspensions, in addition to the active compounds, may contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that may be required. The ointments, pastes, creams and gels may contain, in addition to the compound object of the invention (for example, an isolated or recombinantly produced nucleic acid molecule encoding a LOC387715, SYNPR or PDGFC polypeptide or a LOC387715, SYNPR or PDGFC polypeptide isolated or produced recombinantly), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, bentonites, silicic acid, talc and zinc oxide or mixtures thereof. The powders and sprays may contain, in addition to an object compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. The sprays may also contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. The dosage regimen will be determined by an individual, taking into consideration, for example, several factors that modify the action of the compounds object of the invention, the severity or stage of the AMD, the route of administration and the unique characteristics of the individual, such as age, weight and height. A person with ordinary skill in the art is capable of determining the dosage required to treat the subject. In one embodiment, the dosage may vary from about 1.0 ng / kg to about 100 mg / kg of the subject's body weight. Based on the composition, the dose may be delivered continuously, or at periodic intervals. For example, on one or more separate occasions. The desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art. For example, the compound can be delivered hourly, daily, weekly, monthly, annually (for example, in a temporary release form) or as a one-time supply. In certain embodiments, pharmaceutical compositions suitable for parenteral administration may comprise a LOC387715, SYNPR or PDGFC polypeptide or a nucleic acid molecule encoding a LOC387715, SYNPR or PDGFC polypeptide, in combination with one or more solutions, dispersions, suspensions or emulsions. aqueous or non-aqueous sterile, isotonic, pharmaceutically acceptable, or sterile powders that can be reconstituted in sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the solution isotonic with the intended recipient's blood or agents of suspension or thickeners. Examples of suitable aqueous or non-aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. The compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antimicrobial agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like in the compositions. Adhemase prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. In certain embodiments, the present invention also provides a gene therapy for the in vivo production of the LOC387715, SYNPR or PDGFC polypeptides. Such therapy would achieve its therapeutic effect by introducing the polynucleotide sequences of LOC387715, SYNPR or PDGFC into cells or tissues that are deficient for the normal function of LOC387715, SYNPR or PDGFC. The delivery of the polynucleotide sequences of LOC387715, SYNPR or PDGFC can be achieved using a recombinant expression vector, such as a chimeric virus or a colloidal dispersion system. The selected liposomes can also be used for the therapeutic delivery of the polynucleotide sequences of LOC387715, SYNPR or PDGFC. Various viral vectors that can be used for gene therapy, as taught herein, include adenovirus, herpes virus, vaccinia virus or RNA virus, such as retrovirus. A retroviral vector can be a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted, include non-exclusively: Moloney murine leukemia virus (MoMuLV), Harvey's murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) and Rous Sarcoma Virus (RSV). Several additional retroviral vectors can incorporate multiple genes. All these vectors can transfer or incorporate a gene for a selectable marker, so that the transduced cells can be identified and generated. Retroviral vectors can be made specific for the target by binding, for example, a sugar, a glycolipid or a protein. The preferred selection is achieved using an antibody. Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into a retroviral genome or linked to a viral envelope to allow selection of the specific delivery of the retroviral vector containing the polynucleotide LOC387715, SYNPR or PDGFC. In a preferred embodiment, the vector is directed to the cells or tissues of the eye.

Alternatively, tissue culture cells can be directly transferred with plasmids encoding the retroviral structural genes gag, pol and env, by conventional transfection with calcium phosphate. These cells are then transfected with the plasmid vector containing the genes of interest. The resulting cells release the retroviral vector in the culture medium. Another delivery system selected for the polynucleotides LOC387715, SYNPR or PDGFC is a colloidal dispersion system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and can be delivered to cells in a biologically active form (see, for example, Fraley, et al., Trends Biochem.Sci, 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle are known in the art, see for example, Mannino, et al., Biotechniques, 6: 682, 1988. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The selection of the liposomes is also possible based on, for example, the specificity of the organ, cell specificity and the specificity of the organelle and is known in the art. A person of ordinary skill in the art is capable of determining the amount required to treat the subject. It is understood that the dosage regimen will be determined for an individual, taking into consideration, for example, several factors that modify the action of the compounds object of the invention, the severity or stage of the AMD, route of administration and unique characteristics for the individual, such as age, weight and height. A person with ordinary skill in the art is capable of determining the dosage required to treat the subject. In one embodiment, the dosage may vary from about 1.0 ng / kg to about 100 mg / kg of the subject's body weight. The dose may be given continuously or at periodic intervals. For example, on one or more separate occasions. The desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by someone skilled in the art. For example, the compound can be delivered hourly, daily, weekly, monthly, annually (for example, in a temporary release form) or as a one-time supply. As used herein, the term subject or individual means any animal capable of being afflicted with AMD. Subjects include, but are not limited to, humans, primates, horses, birds, cows, pigs, dogs, cats, mice, rats, guinea pigs, ferrets and rabbits. The samples used in the methods described herein may comprise cells of the eye, ear, nose, teeth, tongue, epidermis, epithelium, blood, tears, saliva, mucus, urinary tract, urine, muscle, cartilage, skin or any other tissue or body fluid from which sufficient DNA or RNA can be obtained. The sample must be processed sufficiently to return to the DNA or RNA that is present, available for testing the methods described herein. For example, the samples can be processed so that the DNA of the sample is available for amplification or for hybridization to another polynucleotide. The processed samples may be used raw where the available DNA or RNA is not purified from other cellular material. Alternatively, samples can be processed to isolate available DNA or RNA from one or more contaminants that are present in their natural source. The samples can be processed by any means known in the art that returns to the available DNA or RNA to test the methods described herein. The methods for processing the samples may include, but are not limited to, mechanical, chemical or molecular means for lysing and / or purifying the cells and those used in cells. Processing methods may include, for example, ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for epitopes. particular of the polypeptide.

Equipment Also provided herein are, for example, equipment for therapeutic purposes or equipment for detecting a LOC387715, SYNPR or PDGFC variant gene in a sample from an individual. In one embodiment, a kit comprises at least one container means having therein placed a pre-measured dose of a polynucleotide probe that hybridizes, under stringent conditions, to a variation in the LOC387715 gene, a variation in the SYNPR gene, or a variation in the PDGFC gene that is correlated with the appearance of AMD in humans. In another embodiment, a kit comprises at least one container means having therein, a pre-measured dose of a polynucleotide primer that hybridizes, under stringent conditions, adjacent to a side of a variation in the LOC387715 gene, a variation in the SYNPR gene, or a variation in the PDGFC gene that is correlated with the appearance of AMD in humans. In a further embodiment, a second polynucleotide primer that hybridizes, under stringent conditions, to the other side of a variation in the LOC387715 gene, a variation in the SYNPR gene, or a variation in the PDGFC gene that is correlated with the appearance of the AMD in humans; it is provided in a pre-measured dose. They may further comprise one or more of a probe or primer, such as one or more of a probe or primer that hybridizes to a variation in LOC387715; one or more of a probe or primer that hybridizes to SYNPR and one or more of a probe or primer that hybridizes to PDGFC. They may further comprise one or more of a probe or primer that hybridizes to a variation in CFH that correlates with AMD and / or one or more of a probe or primer that hybridizes to one or more of the corresponding genes that do not comprise the variation of interest (for example, control or reference genes). The equipment further comprises a mark and / or instructions for the use of therapeutic or diagnostic equipment in the detection of LOC387715, SYNPR or PDGFC in a sample. The equipment also includes packaging material, such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap with heat, bubble wrap, paper, cardboard, starch peanuts, twisted ties, metal fasteners, metal cans, drier desiccant, glass and rubber (see available products from www.papermart.com, for examples of packaging material).

The practice of the present methods will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill in the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2001); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al., Patent of E.U.A. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins, 1984); Transcript And Translation (B. D. Hames &S. Jiggins eds., 1984); Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.), Immunochemical Methods In Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes l-IV (D. M. Weír and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

Exemplification The whole-genome SNP analysis, genotyping the data from 96 cases with AMD and 50 controls without AMD was carried out to identify both the unique associations in LOC387715 and other sites that appear to interact. The following methods and materials have been used in the work described herein.

Haplotype analysis To allocate the haplotypes to 10q26, the Applicants used the version of SNPHAP 1.3 with the default parameters. The inference of the haplotype is subject to errors and therefore, the Applicants also imputed the haplotypes on the same region using the version of PHASE 2.1.1. The two programs use different approaches to estimate haplotypes, and therefore, supposedly would be subject to different errors. Both programs produce identical results in the AREDS data, suggesting exact estimates of the haplotype.

Testing for interactions An appropriate analysis for the interactions of high-dimensional data containing more than 100,000 SNPs can be done in two stages: selecting markers with the statistically significant joint effects, and then modeling the selected markers to quantify the degree of the effects (10) What follows is the procedure of the first stage; The conventional regression analysis (logistic) can be used in the second stage. A simple association test in two sites would involve comparing the frequency of the 9 genotypes with two sites between the cases and the controls. Significant differences could indicate epistasis or major effects of the site. To focus specifically on the interactions, the Applicants first grouped the genotypes of two sites into two or three classes (numbered 1, 2 or 1, 2, 3, respectively) and tested the differences in class frequencies between cases and the controls (Table 1). They used four different classification schemes (Type I-IV) inspired by Cockerham's division of the epistatic variance (11). For each scheme, A already represent the alleles in site 1 (always based on the LOC387715 haplotypes in the data of the Requesters, as described below) and B and b represent the alleles in site 2 (always a SNP in the data). of the Requesters). These classes are defined in Table 1. For each pair of sites to be tested, four independent tests were performed for type I, II, III and IV interactions. For each test, the genotypes of two sites for each individual were recorded in 2 or 3 categories using one of the tables in Table 1. The counts of the genotypic class were then compared between the cases and the controls. Statistical significance was assessed using a Pearson's test 2 with two grades (types I-III) or a degree (type IV) of freedom. Multiple tests were corrected using a Bonferroni correction for 116204 SNP * 4 = 464816 total tests.

Expression analysis Human retinal, placental, kidney and liver samples were obtained from the National Disease Research Interchange (NDRI). The human native RPE and the RPE cultivated human RPE were kindly provided by Dr. Bret Hughes and Dr. Piyoush Kothary, respectively. Total RNA was isolated using TRIZOL (Invitrogen). The first-strand cDNA was generated from 2.5 mg of total RNA, priming with oligo-dT, followed by reverse transcription (www.invitrogen.com). The primers encompassing the templates were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to avoid amplification of the genomic DNA present in the total RNA preparations. . The PCR reactions were adjusted using standard conditions. The expected product sizes are 300 bp for PDFGC, 250 bp for SYNPR and 375 bp for LOC387715. The primers used are as follows: PDGFC-F 5'-GCTGCACACCTCGTAACTTCT-3 '(SEQ ID NO.1) PDGFC-R 5'-GATGCGGCTATCCTCCTGT-3 '(SEQ ID NO 2) SYNPR-F 5'-AAACACTTCTGTGGTCTTTGGA-3' (SEQ ID NO 3) SYNPR-R 5'-AGTGGGGCCAGTAGGCTGT-3 '(SEQ ID NO.4) LOC387715-F d'-TCCCAGCTGCTAAAATCCAC-S '(SEQ ID NO: 5) LOC387715-R 5'-GCTGCACAGAGCAGAAGATG-3' (SEQ ID No. 6) Tissue preparation The eyes of a normal donor were fixed in 4% paraformaldehyde (Grade EM, Polysciences, Warrington, PA) in phosphate buffered physiological saline (PBS) for 6 hours, cryoprotected and included in an optimum short temperature compound (OCT); Miles Laboratory, Elkhart, IN). The frozen sections of the retinas were cut from 8 to 10 μm with a cryostat (Leica microsystem, Bannockbum, IL) and placed on slides (Superfrost / Plus, Fisher Scientific, Fair Lawn, NJ). All human eyes were obtained with the informed consent of the donors, and research with human eyes was conducted in accordance with the principles of the Declaration of Helsinki and the institutional review committee (IRB).

Immunofluorescence microscopy The sections of the retina were blocked for 30 minutes with normal goat serum at (Jackson Immunoresearch, West Grove, PA) diluted in IC buffer (PBS, containing 0.2% Tween-20, 0.1% sodium azide). ) and incubated for 1 hour at room temperature with rabbit anti-rabies synaptophorin antiserum (SYSY, Gottingen, Germany) diluted 1: 50 in staining buffer (IC buffer plus 1% normal goat serum). The sections were washed 3 times in IC buffer and incubated for 1 hour with the 4 'nuclear dye, 6'-diamino-2-phenylindole (DAPI, 1 μg / mL) and Alexa-488 goat anti-rabbit antibodies (Molecular Probes, Eugene, OR) diluted to 1 250 in dye buffer After repeated washing with IC buffer, the sections were covered in mounting medium (Gel Mount, Biomeda, Foster City, CA) and covered with a coverslip For control, the The same concentration of antisinaptopopna antibody was preincubated for 1 hour with the synaptoponne control peptide (SYSY, Gottingen, Germany). The pretreated antibodies were then used to stain the tissue sections as just described. The specimens were analyzed in a confocal microscope with laser scanning (model SP2, Leica Microsystems, Exton, PA). The images of the immunomarked and negative control sections were formed under identical scanning conditions. The images were processed with Photoshop (Adobe Systems, San José, CA) Results The SNP rs10490924 by itself was barely statistically significant in the AREDS data set (both the allelic and genotypic nominal p values are 0 04, Table 2), in part, due to the lower frequency of the allele at risk in the case of the group, compared to the two reports published This difference in frequency may be due to the different definitions of AMD in these studies In the study described here, individuals who had greater than 125 μm in size were required to be the cases (1), whereas in other studies, the pigmentary changes, the neovasculapzation or the geographical atrophy were sufficient for a diagnosis of AMD (8, 9). These observations and additional analysis of the data of the Requesters indicated the existence of other variants that act, in conjunction with SNP rs10490924 to jointly influence the risk of the disease. To explore this further, we defined a set of four SNPs surrounding rs10490924, and showed evidence of ancestral recombination with flanking markers using the four gamete test (FGT). These four SNPs cover approximately 500 nucleotides. Four of the 16 possible haplotypes constituted all the chromosomes in the sample. Two haplotypes were haplotypes of "risk" (N2 and N3), while the other two were haplotypes "without risk" (N1 and N4, Table 3). The two haplotypes at risk are marked by SNP rs2736911 and rs10490924. The difference between the haplotype frequencies of "risk" and "no risk" is statistically significant in cases versus controls (Table 2). The haplotypes imputed for each individual were used to define a new "SNP". For this SNP, an "A" allele means a N1 or N4 haplotype, and a "B" allele means a N2 or N3 haplotype. A genotype of the individual in this "SNP" was assigned based on the haplotypes of the two chromosomes in that individual. A two-way interaction test was performed to examine the differential interactions in cases and controls between this "SNP" derivative (called here 10q26Hap) and all other SNPs in the whole-genome study. Using a strict threshold of Bonferroni p < 0.05 / (4x105), the Requesters obtained two significant results (Pearson's? 2 test, contingency tables in Table 4), which were subsequently duplicated in another population cohort (see below). No significant interaction was found with two SNPs in CH that the Requesters previously found to be associated with AMD. For Y402H, the best interaction had an uncorrected p value of 0.093. For rs380390, the best interaction had an uncorrected p of 0.11. This implies that significant interactions at the Bonferroni threshold may be real and further investigation is warranted. One of these interactions is between SNP rsl0510899 on chromosome 3p14.2 and 10q26Hap; the second is between SNP rs997955 on chromosome 4q32.1 and 10q26Hap. SNPs rsl0510899 and rs997955 are in the sinaptoporin introns (SYNPR) and platelet-derived growth factor C (PDGFC), respectively. Individually, these SNPs do not exhibit a single-site association with AMD in the present study. Notably, these two SNPs are located within two of the six regions with the highest classification that were identified in a recent meta-analysis of studies related to AMD (2). Although the entire genome was explored in this study for interactions in a hypothesis-free manner, the only two significant SNPs at the Bonferroni threshold were located in regions where Applicants hypothesized that they are involved in AMD, based on the previous related studies. In one study, these four genes (CFH, LOC387715, SYNPR and PDGFC) are within four of the main related peaks and an interaction between them has been implicated (12). With the data presented here, a general genetic risk for AMD was estimated. The number of histidine alleles in CFH at position 402 and the number of risk haplotypes in 10q26 in a given individual were added. If the people at risk are defined as those who have a sum of at least two, 82% of the cases would be classified as being at risk, but 48% of the controls would also be classified as being at risk. Instead, the risk was defined based on the genotypes of five SNPs at the four different sites. The overall genetic risk was the sum of three independent risk factors: CFH, the interaction of 10q26Hap with rs10510899 in SYNPR, and the interaction of 10q26Hap with rs997955 in PDGFC. The three possible genotypes or genotypic classes for each risk factor are given a rating that varies from 0 (lower risk) to 2 (higher risk). The sum of these ratings is taken as a measure of overall risk. Individuals with a general rating of 3 or more were considered to be "at risk", while anyone with a rating of 2 or less is "not at risk" (Table 2). With this classification, 81% of cases are at risk, compared to 36% of controls. A risk attributable to the population (PAR) for the effect of this genetic network (Table 2) was estimated to be 71%. Because these interactions are largely derived from the data, there is the possibility of false positives due to the overfitting of Requester data to statistical models for genetic interactions. The best way to assess if there is a real opportunity or association is to duplicate the genotyped SNPs in a second cohort independent of the case and control individuals. DNA was obtained from patients and controls collected at the University of Michigan (6). As with the initial AREDS cohort, all individuals were of European descent to reduce the possibility of false positives due to stratification of the population. In this independent case-control group, SNP rs10490924 is associated to a large extent and significantly with the risk for AMD (Table 2). The frequency of the risk allele observed for the SNP only in the cases is similar to that of the two previous studies, but different from that observed in the AREDS sample. This difference could occur because the Michigan case cohort also included individuals with geographic atrophy and / or neovascularization who did not have large drusen (> 125 μm in diameter), due to subtle differences in the genetic base of the patients in the two studies, or because the Michigan sample was enriched for family cases. Subsequent analyzes show that the two classes of haplotypes in 10q26 are significantly associated with AMD, since there are two interactions identified in the AREDS cohort (Table 2; contingency tables in Table 4). In some cases, the odd ratios for the same risk factor are quite different in the two cohorts. This is probably due to the small size of the sample in the AREDS cohort, and is reflected in the large confidence intervals for this cohort. Additional studies using large cohorts will be necessary to accurately calculate the odd ratios for these risk factors. However, since all reported interactions are statistically significant in the relatively small AREDS cohort and are duplicated independently in the Michigan cohort, the Requesters concluded that these are biologically important interactions. Using the same definition of risk (at least 3 risk factors), the Requesters observed that 63% of the cases were at risk, compared to 32% of the controls (Table 2). For the Michigan sample, the estimated PAR was 55%. The extreme phenotypes in the AREDS study can explain the discrepancy in the PAR between the two samples. Since only 100,000 of the millions of common SNPs in the genome have been genotyped, it is assumed that the two SNPs in SYNPR and PDGFC are probably "branded" SNPs, and functional mutations are other variants located in the genome. To discover functional mutations, genotyping data were examined for a set of ancestor individuals from central and northern Europe in Utah from the HapMap International Project (13). SNP rs10510899 is in link disequilibrium (LD, measured by an appreciable paired r2 correlation) with SNPs spanning approximately 50 kb. This region consists mainly of an intronic sequence for SYNPR, together with a coding exon that does not have a known variant. Only two SNPs among the 100,000 that we genotyped fall into this region. None of these are associated with AMD independently, but exhibit an association in interaction with the 10q26 haplotype. Only the evidence for interaction with rs10510899 exceeds our strict Bonferroni threshold. In the Michigan sample, three additional SNPs near rs10510899 were genotyped to see if they also interact with the 10q26 haplotype. Only one, rs6796563, showed a slightly stronger interaction than rs10510899. This SNP is in the same intron as rs10510899, and is in a weak link disequilibrium with rs10510899 (D -0.37 ^ = 0.05). In contrast, SNP rs997955 is in LD with a larger number of SNPs that span approximately 225 kb. This region includes the intronic sequence of PDGFC, several exon sequences of PDGFC, and the region downstream of the PDGFC gene, but does not overlap with any other known transcribed sequence. Of the 100,000 genotyped, twenty-five formed the map of this region. None is independently associated with AMD, although they show two interactions with the 10q26 haplotype. Among these, the only evidence for interaction that exceeded the strict threshold was with rs997955. In the Michigan sample, four additional SNPs near rs997955 were genotyped. None of these shows a stronger interaction than rs997955. Therefore, functional SNPs associated with AMD appear to reside in the SYNPR and PDGFC genes. To assess whether the three interacting genes (LOC387715, SYNPR and PDGFC) are expressed in the affected target tissue, total RNA from human retinal pigment (RPE), retinal and other tissues was used for RT-PCR analysis. SYNPR transcripts are detected at low levels in the native human RPE, but are expressed largely in the retina and placenta. PDGFC is expressed at high levels in the native human RPE and the cultured RPE, as well as in the retina, placenta and liver. Only a low expression of LOC387715 was observed in the retina and the cultured RPE. Examination of protein expression in eye tissues using commercially available antibodies against sinaptoporin was also performed. The inner plexiform layer showed the strongest mark for sinaptophorin antibodies; however, the outer plexiform layer was also marked, although the signal is weaker. This is consistent with the previously reported location of sinaptoporin in the rabbit retina (14). The distribution of synaptophorin in the presynaptic terminals in the horizontal cells is involved in the release of the synaptic vesicles. Since these cells provide an inhibitory input that contributes to the antagonistic responses that surround the center of the bipolar neurons, altering the efficiency of this input could lead to abnormal levels of photoreceptor synaptic activity and consequent cell damage. PDGFC is part of the regulatory cascade that controls the activity of the matrix metalloproteinases and their tissue inhibitors, the molecules intimately involved in regulating vascular quiescence and growth in the eye, as well as in other tissues (15). The interaction of LOC387715 with two putatively unrelated genes, SYNPR and PDGFC, suggests a common regulatory function in the retina or RPE. Preliminary studies indicate that the distribution of PDGFC in the inner nuclear layer and the cells of the normal retina ganglia (data not shown); neither SYNPR nor PDGFC is detectable in the positive druse of CFH. To provide a general understanding of the invention, certain illustrative modalities are described, including compositions and methods to identify or help identify individuals at risk of developing AMD, as well as to diagnose or assist in the diagnosis of AMD. However, it will be understood by one of ordinary skill in the art that the compositions and methods described herein may be adapted and modified as appropriate for the application to be treated, and that the compositions and methods described herein may be employed in others. appropriate applications, and that such other additions and modifications will not depart from the scope of the same.

References 1. Klein RJ, Zeíss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005; 308 (5720): 385-9. 2. Fisher SA, Abecasis GR, Yashar BM, et al. Meta-analysis of genome scans of age-related macular degeneration. Hum Mol Genet 2005; 14 (15): 2257-64. 3. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant ncreases the risk of age-related macular degeneration. Science 2005; 308 (5720): 419-21. 4. Edwards AO, Ritter R, 3rd, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H polymorphism and age-related macular degeneration. Science 2005; 308 (5720): 421-4. 5. Hageman GS, Anderson DH, Johnson LV, et al. A common haplotype in the regulatory complement gene factor H (HF1 / CFH) predisposes individuáis to age-related macular degeneration. Proc Nati Acad Sci USA 2005; 102 (20): 7227-32. 6. Zareparsi S, Branham KE, Li M, et al. Strong Association of the Y402H Variant in Complement Factor H at 1q32 with Susceptibility to Age-Related Macular Degeneration. Am J Hum Genet 2005, 77 (1): 149-53. 7. Conley YP, Thalamuthu A, Jakobsdottir J, et al. Candidate gene analysis suggests a role for fatty acid biosynthesis and regulation of the complement system in the etiology of age-related maculopathy. Hum Mol Genet 2005. 8. Rivera A, Fisher SA, Fritsche LG, et al. Hypothetical LOC387715 is a second major susceptibility gene for age-related macular degeneration, contributing independently of complement factor H to disease risk. Hum Mol Genet 2005; 14 (21): 3227-36. 9. Jakobsdottir J, Conley YP, Weeks DE, Mah TS, Ferrell RE, Gorin MB. Susceptibility genes for age-related maculopathy on chromosome 10q26. Am J Hum Genet 2005; 77 (3): 389-407. 10. Hoh J, Wille A, Zee R, et al. Selecting SNPs in two-stage analysis of disease association data: a model-free approach. Ann Hum Genet 2000; 64: 413-7. 11. Cockerham CC. An Extension of the Concept of Partitioning Hereditary Variance for Analysis of Covariates Among Relativs When Epistasis is Present. Genetics 1954, 39: 859-82. 12. Majewski J, Schultz DW, Weleber RG, et al. Age-related macular degeneration- a genome sean in extended families. Am J Hum Genet 2003; 73: 540-50. 13. Altshuler D, Brooks LD, Chakravarti A, Collins FS, Daly MJ, Donnelly P. A haplotype map of the human genome. Nature 2005, 437 (7063): 1299-320. 14. Brandstatter JH, Lohrke S, Morgans CW, Wassle H. Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, n the mammalian retina. J Comp Neurol 1996; 370 (1): 1-10. 15. Li X, Ponten A, Aase K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol 2000; 2 (5): 302-9. 16. Seddon JM, Cote J, Page WF, Aggen SH, Neale MC. The US twin study of age-related degenerative macular: relative roles of genetíc and environmental nfluences. Arch Ophthalmol 2005; 123 (3): 321-7.

TABLE 1 Definitions of the four classes of epistasis TABLE 2 Evidence of association and odd relationships (OR). Note that the risk factor for calculating the ORs may be the combination of two classes of the y2 tests. Population A is the AREDS sample; M is the Michigan sign Population Try 1 N Value of Df Factor of OR (Cl of P 2 risk 95%) A rs10490924 146 0041 2 2 At least one 3- (1 1 -4 6) risk allele A 146 0 0 018 Class 2 At least one 3 7 (1 8-7 9) haplotype risk haplotype A 10q26Hap X 146 1 3e -08 2 Class of 2 2 (6 2-8 1) ts10510899 (medium risk type I) or high A 10q26Hap x rs 140 4 5e-08 2 Class of 9 1 (4 0-2 2) 997955 (type III) medium or high risk A Sum of the factor 136 1 1 e-07 1 Sum > = 3 7 9 (5 to 1 March 8) nsk M rs10490924 367 1 1 e-08 2 At least 3 2- (2 0-4 8) risk allele M classes 367 0 000 037 2 At least one August 1 ( 1 September 2-2) haplotype risk haplotype M 10q26Hap x 365 0017 2 Class of June 1 (1 0-2 7) rs10510899 (medium risk type I) or high M 10q26Hap x rs 357 0 00 044 2 Class August 1 ( 1 1 -7 8) 997955 (type III) medium or high risk M Sum of factor 345 1 2e-08 1 Sum > = 3 3 7 (2 3-5 8) of risk TABLE 3 Haplotypes surrounding rsl 0490924. The y2 statistic for these 292 observed haplotypes is 12.5. With 3 degrees of freedom, this translates to a p-value of 0.006 Haplotype rs 10490922 rs 10490923 rs2736911 rs 10490924 Case Control N1 A A C G 28 13 N2 T G T G 32 12 N3 T G C T 68 21 N4 T G C G 64 54 TABLE 4 Counts of the genotype observed for 10q26Hap. rsl 0510899 and rs997955. The counts are provided for each of the nine pairs of possible genotypes AREDS 10q26Hap Cases Controls AA AB BB Total AA AB BB Total rs10510899 AA 3 32 14 49 21 1 1 3 35 AB 15 17 9 41 2 6 4 12 BB 1 5 0 6 1 2 0 3 Total 19 54 23 96 24 19 7 50 CD rs997955 AA 1 1 45 20 76 23 15 3 41 00 AB 8 7 1 16 0 2 4 6 BB 0 0 0 0 1 0 0 1 Total 19 52 21 92 24 17 7 48 Michigan 10q26Hap Cases Controls AA AB BB Total AA AB BB Total rs10510899 AA 26 50 36 1 12 42 52 14 108 AB 18 26 23 67 18 24 10 52 BB 1 3 6 10 4 10 2 16 Total 45 79 65 189 64 86 26 176 rs997955 AA 34 65 56 155 55 69 22 146 AB 8 9 7 24 9 15 4 28 BB 0 2 0 2 1 1 0 2 Total 42 76 63 181 65 85 26 176 CD CD

Claims (21)

NOVELTY OF THE INVENTION CLAIMS

1. A composition for treating a subject suffering from, or at risk of, age-related macular degeneration, comprising: (a) an effective amount of (1) a wild type, isolated or produced LOC387715 polypeptide recombinant, or a fragment thereof; (2) a wild-type SYNPR polypeptide, isolated or recombinantly produced, or a fragment thereof; or (3) a PDGFC polypeptide of the wild type, isolated or recombinantly produced, or a fragment thereof and (b) a pharmaceutically acceptable carrier.

2. The composition according to claim 1, further characterized in that it comprises an effective amount of a wild type, isolated or recombinantly produced CFH polypeptide or a fragment thereof.

3. The use of the composition of claim 1 or 2, for the manufacture of a medicament useful for treating a subject suffering from or at risk of age-related macular degeneration.

4. A composition for treating a subject suffering from, or at risk of, age-related macular degeneration, comprising: (a) an effective amount of (1) an isolated or produced nucleic acid molecule recombinant, which encodes a LOC387715 polypeptide, or a fragment thereof; (2) an isolated or recombinantly produced nucleic acid molecule encoding a SYNPR polypeptide, or a fragment thereof; or (3) an isolated or recombinantly produced nucleic acid molecule encoding a PDGFC polypeptide, or a fragment thereof; and (b) a pharmaceutically acceptable carrier.

5. The use of the composition of claim 4, for the manufacture of medicament useful for treating a subject suffering from or at risk of age-related macular degeneration.

6. A composition for treating a subject suffering from, or at risk of age-related macular degeneration, comprising: (a) a nucleic acid molecule comprising an antisense sequence that hybridizes to (i) a LOC387715 variant gene or mRNA, which is correlated with the onset of age-related macular degeneration in humans, (ii) a variant SYNPR gene or mRNA that is correlated with the onset of age-related macular degeneration in humans or (iii) a variant PDGFC gene or mRNA that is correlated with the onset of age-related macular degeneration in humans; and (b) a pharmaceutically acceptable carrier.

The use of the composition of claim 6, for the manufacture of a medicament useful for treating a subject suffering from, or at risk of, age-related macular degeneration.

8. A composition for treating a subject suffering from, or at risk of, age-related macular degeneration, comprising: (a) a nucleic acid molecule comprising a siRNA or mRNA sequence, or a precursor of the same, that hybridizes to (i) a LOC387715 variant gene or mRNA that is correlated with the onset of age-related macular degeneration in humans; (ii) a variant SYNPR gene or mRNA that is correlated with the onset of age-related macular degeneration in humans; or (iii) a variant PDGFC gene or mRNA that is correlated with the onset of age-related macular degeneration in humans; and (b) a pharmaceutically acceptable carrier.

9. The use of the composition of claim 8 for the preparation of a medicament useful for treating a subject suffering from or at risk of age-related macular degeneration.

10. The use as claimed in claims 3, 5, 7 or 9, further comprises, before the step of administering said medicament, detecting the presence or absence of a risk variation in the polymorphic site LOC387715 in a sample of the patient, where the presence of a risk variation in the polymorphic site LOC387715 indicates that the patient has or is at risk of developing AMD.

11. An isolated polynucleotide for the detection of a variant gene that is correlated with the appearance of age-related macular degeneration in humans, comprising a nucleic acid molecule selected from the group consisting of: (a) a molecule of nucleic acid that specifically detects a variation in the LOC387715 gene, which is correlated with age-related macular degeneration in humans; (b) a nucleic acid molecule that specifically detects a variation in the SYNPR gene, which is correlated with the onset of age-related macular degeneration in humans; and (c) a nucleic acid molecule that specifically detects a variation in the PDGFC gene, which is correlated with the onset of age-related macular degeneration in humans.

12. The polynucleotide according to claim 11, further characterized in that the polynucleotide is a probe that hybridizes, under stringent conditions, to a variation selected from the group consisting of: (a) a variation in the LOC387715 gene, which is correlated with the appearance of macular degeneration related to age in humans; (b) a variation in the SYNPR gene, which is correlated with the onset of age-related macular degeneration in humans; and (c) a variation in the PDGFC gene, which is correlated with age-related macular degeneration in humans.

13. The polynucleotide according to claim 12, further characterized in that the variation encodes an amino acid other than alanine at position 69 of the LOC387715 protein.

14. - The polynucleotide according to claim 13, further characterized in that the variation encodes the serine at position 69 of the LOC387715 protein.

15. A diagnostic device for detecting a variant gene correlated with age-related macular degeneration in a sample of an individual, comprising: the polynucleotide of the claim eleven; a container, and a label and / or instructions for the use of the diagnostic equipment in the detection of variant genes in a sample.

16. The diagnostic equipment according to claim 15, further characterized in that it comprises a polynucleotide probe that hybridizes, under stringent conditions, to a variation in a CFH gene that is correlated with the onset of age-related macular degeneration. .

17. A method for identifying an individual at risk of developing age-related macular degeneration, comprising detecting the presence of a variant gene or polypeptide that is correlated with the onset of age-related macular degeneration in humans, wherein the variant gene or polypeptide is selected from the group consisting of: a variant LOC387715 gene or polypeptide; a variant SYNPR gene or polypeptide and a variant PDGFC gene or polypeptide, and wherein the presence of the variant gene or polypeptide indicates that the individual is at risk of developing age-related macular degeneration.

18. - The method according to claim 17, further characterized in that the variant encodes a different amino acid of alanine at position 69 of the LOC387715 protein.

19. The method according to claim 18, further characterized in that the variant encodes the serine in position 69 of the LOC387715 protein.

20. The method according to claim 17, further characterized in that the detection step comprises: (a) combining a sample obtained from the individual with (1) a polynucleotide probe that hybridizes, under stringent conditions, to a variation in the LOC387715 gene, which is correlated with the occurrence of age-related macular degeneration in humans, but does not hybridize to a LOC387715 gene that does not contain the variation; (2) a polynucleotide probe that hybridizes, under stringent conditions, to a variation in the SYNPR gene, which is correlated with the onset of age-related macular degeneration in humans, but does not hybridize to a SYNPR gene that does not contains the variation; and (3) a polynucleotide probe that hybridizes, under stringent conditions, to a variation in the PDGFC gene, which is correlated with the onset of age-related macular degeneration in humans, but does not hybridize to a PDGFC gene that it does not contain the variation; and (b) determining if hybridization occurs, where the occurrence of the hybridization of the probes of (a) (1) - (a) (3) indicates that the individual is at risk of developing macular degeneration related to age.

21. - The method according to claim 17, further characterized in that it comprises detecting the presence of a variant CFH gene that correlates with the onset of age-related macular degeneration.