MXPA00010172A - Novel mutations in the freac3 - Google Patents

Abstract

The invention features novel mutations in the FREAC3 gene. Our discovery provides methods for early diagnosis of glaucoma, other disorders of the eye, and heart defects. Also provided are cells having at least one deficient FREAC3 gene. Such cells may be used to detect therapeutic compounds that mimic FREAC3, are agonists of FREAC3, or otherwise modulate the level of FREAC3 biological activity.

Description

NOVEDOUS MUTATIONS IN THE GENE FREAC3 FOR DIAGNOSIS AND PROGNOSIS OF GLAUCOMA AND PREVIOUS SEGMENT DISGENESIS • Background of the Invention Glaucoma, a major cause of blindness worldwide, is characterized by the progressive degeneration of the optic medium which is usually associated with increased intraocular pressure. In most cases, blindness from glaucoma begins with loss of peripheral vision. The central vision is maintained until the last stage of the disease. When visual loss is noted, the damage is advanced. Although glaucoma has a frequency of occurrence as high as that of high blood pressure and diabetes, the lack of warning to the public results in thousands of new cases of blindness occurring annually. For example, the Canadian National Institute for the Blind has identified glaucoma as one of the two leading causes of blindness in Canada. And in the United States, more than 1.2 million people have vision loss and more than 80,000 people are legally blind as a result of glaucoma. Fortunately, most cases of glaucoma can be treated successfully, and vision loss can be avoided by using existing drugs or surgical approaches. The key to the successful treatment of glaucoma lies in its early detection, before irreversible damage to the optic nerve has occurred. The dysgenesis of the anterior segment, the incorrect formation of structures of the anterior segment of the eye, underlies the pathogenesis of some cases of congenital glaucoma. Glaucoma in patients with anterior segment dysgenesis is probably a result of incorrect regulation of outflow of aqueous humor due to inadequate development of anterior segment angle structures. Several autosomal dominant disorders of anterior segment formation that result in glaucoma have recently been genetically colocalized on chromosome 6p25. These disorders include iridogonium-dysgenesis abnormality (AGID), Axenfeld-Rieger anomaly (ARA), familial glaucoma iridogoniodis-plasia (FGI), and familial glaucoma with goniodisgenesis. Since glaucoma is the leading cause of blindness worldwide, it would be desirable to have a simple diagnostic test to identify those with increased risk of blindness due to glaucoma. It would also be desirable to have experimental assays and animal models for the identification of compounds that are useful for the prevention or treatment of glaucoma. Compendium of the Invention The discovery of mutations in the FREAC3 genes of patients with IRID1 has been reported here. The FREAC3 gene is a member of the gene family of hairpin transcription factor / winged helix. The results of mouse expression and analysis studies with homozygous clippings of Mfl, the murine homologue of FREAC3, are consistent with a role for Mfl / FREAC3 in eye development and glaucoma. The diagnosis of the genetic defect in the IRID1 family, as provided herein, will allow the immediate monitoring and presymptomatic treatment of glaucoma that IRID1 patients frequently develop. Moreover, mutations of the IRID1 gene may be responsible for a significant portion of glaucoma patients not clinically diagnosed with IRID1, since not all patients with IRID1, even with IRID1 families, have iris defects otherwise used to diagnose the IRID1. The discovery provides methods for the early diagnosis of glaucoma and other disorders in the eye. Also provided are cells that have at least one FREAC3 gene deficient. These cells can be used to detect new therapeutic compounds that mimic FREAC3, are FREAC3 agonists, or otherwise increase the level of biological activity of FREAC3. In a first aspect, the invention presents a method of diagnosing a mammal by an increased likelihood of developing an eye disease, which comprises analyzing the nucleic acid of the mammal to determine if the nucleic acid contains a mutation in a FREAC3 gene. The presence of a mutation is an indication that the mammal has an increased likelihood of developing an eye disease. In a second aspect, the invention presents a method for diagnosing a mammal by an increased probability of having a developmental defect, which comprises analyzing the nucleic acid of the mammal to determine whether the carbon atom contains a mutation in a FREAC3 gene. The presence of a mutation is an indication that the mammal has an increased likelihood of having a developmental defect. In preferred embodiments of this second aspect of the invention, the developmental defect is a cardiac defect or is the dysgenesis of the anterior segment. In a preferred embodiment of the first and second aspects of the invention, the mutation is a nonsense mutation. For example, the nonsense mutation may be a transverse of G to C in the coding nucleotide 245, which results in a Ser82Thr mutation in the helix 1 of the FREAC3 hairpin domain, or the mutation without sense may be a transversion from G to C in coding nucleotide 261, which results in an Ile87Met mutation in helix 1 of the hairpin domain of FREAC3. In another modality of the first and second aspects, the mutation may be a mutation of phase shifting. For example, the frame shift mutation may result from a deletion of 10 base pairs of coding nucleotides 93 through 102. A frame shift mutation may result in a truncated protein. In other embodiments of the first and second aspects, primers can be used to detect the mutation, these primers can be selected from those shown in Table 1. The methods of the first and second aspects can further comprise the step of sequencing the nucleic acid which encodes FREAC3 from the mammal. In addition, the methods may further comprise the step of using nucleic acid primers specific for the FREAC3 gene, which is used for the amplification of DNA by the polymerase chain reaction. In yet other embodiments of the first and second aspects, analyzes include detecting the loss of a recognition site for a restriction endonuclease (eg, Alu I), or the analysis includes detecting the gain of a recognition site for an endonuclease of restriction (for example, Bsp Hl). The analysis may also include detecting a loss of one or more nucleotides, or a gain of one or more nucleotides. In addition, the analysis may include detection that does not match, using single-strand conformation polymorphism analysis (SSCP), or restriction fragment length polymorphism (RFLP) analysis. In a third related aspect, the invention presents a set for the analysis of FREAC3 nucleic acid. The kit comprises nucleic acid probes for analyzing the nucleic acid of a mammal, wherein the analysis is sufficient to determine whether the mammal contains a mutation in the FREAC3 nucleic acid. In a fourth aspect, the invention features a method for making an antibody that specifically binds a mutant FREAC3 polypeptide, comprising administering a mutant FREAC3 polypeptide, or fragment thereof, to an animal capable of generating an immune response, and isolating the antibody of the animal. In a fifth aspect, the invention features a method for detecting the presence of a mutant FREAC3 polypeptide, comprising contacting a sample with an antibody that specifically binds to a mutant FREAC3 polypeptide and testing for binding of the antibody to the polypeptide. mutant In the preferred embodiments of the fifth aspect, the mutant FREAC3 polypeptide can have a threonine residue at amino acid position 82 of FREAC3 3, or a methionine residue at amino acid position 87 of FREAC3 3, or mutant FREAC3 3 polypeptide can have an amino acid sequence that differs from the sequence of the natural type of FREAC3 where the amino acid sequence that differs is carboxyterminal to amino acid 33 of FREAC3 (wing 33). In a sixth aspect, the invention features a method of diagnosing a mammal by an increased likelihood of developing an eye disease, which comprises detecting the presence of a mutant FREAC3 polypeptide in the mammal. The presence of a mutant FREAC3 polypeptide indicates that the mammal has a mutation in a FREAC3 gene, and the presence of a mutation is an indication that the mammal has an increased likelihood of developing an eye disease. In a seventh aspect, the invention features a method of diagnosing a mammal by an increased probability of having a developmental defect, which comprises detecting the presence of a mutant FREAC3 polypeptide in the mammal. The presence of a mutant FREAC3 polypeptide indicates that the mammal has a mutation in a FREAC3 gene 3, and the presence of a mutation is an indication that the mammal has an increased likelihood of having a developmental defect. In an eighth aspect, the invention presents a set for FREAC3 nucleic acid analysis, comprising antibodies for analyzing polypeptides of a mammal, wherein the analysis is sufficient to determine if the mammal contains a mutation of FREAC3 nucleic acid . In a ninth aspect, the invention features nucleic acid encoding FREAC3 mutant. The nucleic acid has at least one mutation, and the mutation is an indication that a mammal from which the nucleic acid is derived has an increased likelihood of developing glaucoma. • In various embodiments of the ninth aspect of the invention, the mutation may be a transversion from G to C in the coding nucleotide 245, or a transversion from G to C in the coding nucleotide 261, or a deletion of the coding nucleotides. 93 through 102. In another embodiment, the nucleic acid is operably linked to the regulatory W sequences for the expression of FREAC3, and the regulatory sequences comprise a regulator. In preferred embodiments of the first, second, third, sixth, seventh, eighth and ninth aspects of the invention, the mammal is human, or the mammal is prenatal. In a tenth aspect, the invention features a cell containing the nucleic acid of the ninth aspect of the invention. In other embodiments of the tenth aspect of the invention, the cell can be a prokaryotic cell, a eukaryotic cell, such as a yeast cell or a mammalian cell. In another embodiment of the tenth aspect, the promoter may be inducible. In an eleventh aspect, the invention features a non-human transgenic mammal that contains the nucleic acid of the ninth aspect of the invention. The nucleic acid is operatively linked to the regulatory sequences for the expression of FREAC3. In a preferred embodiment of the eleventh aspect, the mammal can be a rodent. In another preferred embodiment, one or both of the endogenous alleles encoding a FREAC3 polypeptide can be disrupted, suppressed, or otherwise rendered non-functional. In a twelfth related aspect, the invention features cells of the transgenic mammal of the tenth aspect of the invention. In a thirteenth aspect, the invention characterizes a non-human mammal in which one or both of the endogenous alleles encoding the FREAC3 polypeptide are mutated in the positions corresponding to those shown in Figure 2. In a fourteenth related aspect, the invention features cells of the mammal of the thirteenth aspect of the invention. In a fifteenth aspect, the invention features a method for detecting a compound useful for the prevention or treatment of an eye disease., which comprises testing the transcription levels of a reporter gene operably linked to a promoter, wherein the promoter contains a FREAC3 binding site. The method includes the steps of: (a) exposing the reporter gene to the compound, and (b) testing the reporter gene to determine an alteration in the reporter gene's activity in relation to the reporter gene not being exposed to the compound.

In various embodiments of the fifteenth aspect of the invention, the reporter gene may be in a cell, the cell may be in an animal, and an increase in transcription indicates • a compound useful for the prevention or treatment of glaucoma. In a preferred embodiment of the first, sixth, and fifteenth aspects of the invention, the eye disease is glaucoma. In a sixteenth aspect, the invention presents a method for treating a disease in the eye by means of • live gene, which comprises introducing a nucleic acid encoding FREAC3 of the wild type into the cells of the eye. The nucleic acid is operably linked to regulatory sequences for the expression of FREAC3, the regulatory sequences comprise a promoter, and the expression of FREAC3 is sufficient to improve the symptoms of the disease. In preferred embodiments of the sixteenth aspect, the nucleic acid can be introduced into the cells by means of a viral vector, which contains the nucleic acid encoding the FREAC3 or the nucleic acid can be introduced into the 0 cells by transformation. By "FREAC3 nucleic acid" or "FREAC3 gene" is meant a nucleic acid, such as a genomic DNA, cDNA, or mRNA, encoding FREAC3, a FREAC3 protein, FREAC3 polypeptide, or portion thereof, as defined below . A FREAC3 nucleic acid can also be a FREAC3 primer or probe, or antisense nucleic acid that is complementary to the mRNA encoding FREAC3. By "FREAC3 wild type" is meant a FREAC3 nucleic acid or FREAC3 polypeptide having the nucleic acid or amino acid sequence most frequently observed in the members of a given animal species and not associated with a disease phenotype. The wild type FREAC3 is FREAC3 biologically active. A wild-type FREAC3 is, for example, a FREAC3 polypeptide or nucleic acid having the sequence shown in Figure 2. The wild-type FREAC3 can also be a polymorphic FREAC3 as described herein (ie, insertion of a codon). extra GGC (glycine) after coding nucleotide 345 or 447, as described below). By "FREAC3 mutant" "FREAC3 mutation or mutations" or "FREAC3 mutations" is meant FREAC3 polypeptide or nucleic acid having a sequence that deviates from the wild-type sequence in a manner sufficient to confer an increased probability of developing the malformations of the anterior segment and / or glaucoma in at least some genetic and / or environmental antecedents. These mutations can occur naturally, or can be induced artificially. They can be, without limitation, insertion, suppression, phase shifting, or nonsense mutations; these mutations may result in replacement of the wild-type amino acid with a different amino acid, or premature termination of the polypeptide.

A mutant FREAC3 protein may have one or more mutations, and these mutations may affect various aspects of the biological activity of FREAC3 (protein function), to various degrees. Alternatively, a FREAC3 mutation can indirectly affect the biological activity of FREAC3 by influencing, for example, the transcription activity of a gene encoding FREAC3, or the stability of FREAC3 mRNA. For example, a mutant FREAC3 gene may be a gene expressing a mutant FREAC3 protein or it may be a gene that alters FREAC3 protein level in a sufficient manner to confer an increased statistically significant probability (p = 0.05) of developing glaucoma at less a genetic and / or environmental background. By "FREAC3 biologically active" it is understood that FREAC3 within an individual is sufficient to prevent anterior segment dysgenesis or the development / progression of FREAC3-dependent glaucoma in an individual by everything else. An assessment of the relative biological activity of FREAC3 can be made in an individual, for example, by comparing the FREAC3 sequence in the individual to know the FREAC3 sequences of the wild type and mutants, by measuring the relative amount of the FREAC3 link in a sample to a FREAC3 binding site (eg, aGTAAA (T / c) AAAca), or by measuring the relative capacity of FREAC3 in a sample to transactivate the expression of a FREAC3-dependent gene (for example, by measuring the expression of the reporter gene from a chimeric gene containing a FREAC3 binding site in its regulatory region), in relation to that of FREAC3 of the wild type, or • through equivalent approaches. The prevention and / or treatment of glaucoma can be carried out by increasing the biological activity of a FREAC3 molecule or by increasing the number of FREAC3 molecules in a patient with relatively low FREAC3 biological activity. Preferably, the biological activity of FREAC3 is at least 25 percent that of a normal individual, more preferably, at least 50 percent, still more preferably, at least 75 percent, and much more preferably, at least 90 percent than in a normal individual. By "Mfl" we mean the mouse counterpart of human FREAC3. The definitions presented above for FREAC3 (eg, "natural type", "mutated") apply to Mfl. By "endogenous allele" is meant a copy of a gene that has not been artificially inserted into a genome. By "mutation without sj = ntido" is meant the substitution of a purine or pyrimidine base (ie, A, T, G, or C) by another with a nucleic acid sequence so that the new codon results in a different amino acid. of the amino acid originally encoded by the network by the reference codon (for example, the wild type). By "frame-shifted mutation" is meant the insertion or deletion of at least one nucleotide within a polynucleotide coding sequence. A mutation of frame shift alters the reading frame of the codon and / or • downstream of the mutation site. This mutation results in either the replacement of the wild-type amino acid sequence encoded by a novel amino acid sequence, or a premature termination of the encoded polypeptide due to the creation of a stop codon, or both. By "high priority conditions" are meant 1W conditions that allow hybridization comparable to hybridization that occurs during overnight incubation using a DNA probe of at least 500 nucleotides in length, in a solution containing 0.5 M NaHP04, pH 7.2, 7 percent SDS, 1 mM EDTA, 1 percent BSA (fraction V), and 100 micrograms / milliliter of denatured, sheared salmon sperm DNA at a temperature of 65 ° C, or a solution containing 48 percent formamide, 4.8X SSC (150 mM NaCl, 15 mM • trisodium citrate), 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10 percent dextran sulfate, 0.1 percent SDS, and 100 micrograms / milliliter of denatured, sheared salmon sperm DNA at a temperature of 42 ° C (these are typical conditions for high Northern or Southern, or colony high priority hybridizations). High priority hybridization can be used for techniques such as chain reaction of High priority polymerase, DNA sequencing, single chain conformation polymorphism analysis, and site hybridization. The aforementioned techniques are usually performed with relatively short probes (eg, usually 16 nucleotides or longer for polymerase chain reaction or sequencing, and 40 nucleotides or longer for site hybridization). The high priority conditions used in these techniques are well known to those skilled in the art of molecular biology, and can be found, for example, in FREAC3 Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1997, incorporated herein by reference. By "analyzing" or "analysis" is meant subjecting a FREAC3 nucleic acid or a FREAC3 polypeptide to a test procedure that allows the determination of whether the FREAC3 gene is of the natural or mutant type. For example, the FREAC3 genes of an animal can be analyzed by amplifying the genetic DNA using the polymerase chain reaction, and then determining the DNA sequence of the amplified DNA. To "prove" means to analyze the effect of a treatment or exposure, either chemical or physical, administered to the whole animal or to cells derived from it. The material being analyzed can be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting altered gene expression, altered nucleic acid stability (e.g., mRNA stability), altered protein stability, altered protein levels, or altered protein biological activity. The means to analyze can • include, for example, nucleic acid amplification techniques, reporter gene assays, antibody labeling, immunoprecipitation, and phosphorylation assays, and other techniques known in the art to carry out the analysis of the invention. By "modulation" is meant to change, either decreases • going or increasing. By "probe" or "primer" is meant a single-stranded DNA molecule or RNA of defined sequence so that it can be paired by base with a second DNA or RNA molecule containing a complementary sequence (the "target") . The stability of the resulting hybrid depends on the extent of base mating that occurs. The extent of base pairing is affected by parameters such as the degree of complementarity between the probe and the target molecules, and the degree of priority of the hybridization conditions. Hybridization priority grade 0 is affected by parameters such as temperature, salt concentration, and concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes and primers specific for the FREAC3 nucleic acid will preferably have at least a sequence identity of 35 percent, more preferably at least a sequence identity of 45-55 percent, still more preferably at least one sequence identity at 60-75 percent, • still more preferably at least one sequence identity of 80-90 percent, and more preferably 100 percent sequence identity. The probes can be detectably labeled, either radioactively or non-radioactively, by methods well known to those skilled in the art. The probes are used for methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by polymerase chain reaction, single-strand conformation polymorphism analysis (SSCP), restriction fragment polymorphism analysis (RFLP), Southern hybridization, Northern hybridization, site hybridization, electrophoretic mobility shift (EMSA) assay. By "mismatch detection approach" is meant the identification of a mutation (i.e., a mismatch) in a gene using standard techniques to analyze the nucleic acid of a patient sample. Generally, these techniques involve amplification by nucleic acid polymerase chain reaction of the patient's sample, followed by identification of the mutation either by altered hybridization, aberrant electrophoretic gel migration, linkage or dissociation mediated by missing link proteins. coincidence, direct nucleic acid sequencing, or other techniques that are known in the art. By "pharmaceutically acceptable carriers" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. An exemplary pharmaceutically acceptable vehicle is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mach Publishing Company, Easton, PA. By "identity" means that a sequence of polypeptides or nucleic acids possess the same amino acid or nucleotide residue at a given position, as compared to a reference polypeptide or nucleic acid sequence to which the first sequence is aligned. Sequence identity is typically measured using sequence analysis analysis software with the default parameters specified therein, such as the introduction of gaps to achieve optimal alignment (eg, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WL 53705 ). This software program pairs similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50 percent, preferably 85 percent, more preferably 90 percent, and still more preferably 95 percent identity with an amino acid or reference nucleic acid sequence . For polypeptides, the length of the comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and still more preferably 35 amino acids. For nucleic acids, the length of the comparison sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and still more preferably 110 nucleotides. By "substantially pure polypeptide" is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60 percent, by weight, free of naturally occurring proteins and organic molecules with which it naturally associates. Preferably, the polypeptide is a FREAC3 polypeptide that is at least 75 percent, more preferably at least 90 percent, and still more preferably at least 99 percent, by weight, pure. A substantially pure FREAC3 polypeptide can be obtained, for example, by extracting a natural source (e.g., a peripheral blood leukocyte), by expressing a recombinant nucleic acid encoding a FREAC3 polypeptide, or by chemically synthesizing the protein. The purity can be measured by a suitable method, for example, by chromatography column, polyacrylamide gel electrophoresis, or high performance liquid chromatography analysis. A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced a cellular system different from the cell from which it naturally originates will be substantially free of its naturally associated components. In accordance with the foregoing, substantially pure polypeptides include not only those derived from eukaryotic organisms but also those synthesized in E. coli or other prokaryotes.

By "substantially pure DNA" is meant DNA that is free from genes which, in the naturally occurring genome of the organism from which the DNA of the invention is derived, flank the gene. Thus the term includes, for example, a recombinant DNA that is incorporated into a vector; in a plasmid or autonomously replicating virus; or in the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid that encodes an additional polypeptide sequence. By "transgene" is meant any piece of DNA that is artificially inserted into a cell, and becomes part of the genome of the organism which develops from that cell. This transgene may include a gene that is partially or completely heterologous (ie, foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. By "transgenic" is meant any cell that includes a DNA sequence that is artificially inserted into a cell and becomes part of the genome of the organism that develops from that cell. As used herein, transgenic organisms are generally transgenic mammals (eg, rodents such as rats or mice) and DNA (transgene) is artificially inserted into the nuclear genome. By "clipping mutation" is meant an alteration in • the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80 percent relative to the non-mutated gene. The mutation may, without limitation, be an insertion, deletion, mutation of frame shift, or nonsense mutation. Preferably, the mutation is an insertion or deletion, or p is a frame shift mutation that creates a stop codon. By "transformation" is meant any method for introducing foreign molecules into a cell. Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral administration, electroincorporation, and biolistic transformation are only some of the methods known to those skilled in the art that can be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity-driven microprojectiles such as tungsten or gold particles. These velocity-driven methods originate from pressure explosions that include, but are not limited to, helium-driven, air-driven techniques and gunpowder-driven techniques. The biolistic transformation can be applied to the transformation or transfection of a wide variety of intact cell types and tissues including, without limitation, intracellular organelles (eg, mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells. By "transformed cell" is meant a cell in which (or within an ancestor of which) a DNA molecule encoding (as used herein) has been introduced, by means of recombinant DNA techniques, a polypeptide FREAC3. By "positioned for expression" is meant that the DNA molecule is placed adjacent to a DNA sequence that directs the transcription and translation of the sequence (i.e., facilitates the production of, for example, a FREAC3 polypeptide, a recombinant protein or an RNA molecule). By "promoter" is meant a minimum sequence sufficient to direct the transcription. Also included in the invention are those promoter elements which are sufficient to render the expression of the promoter-dependent gene controllable for specific cell-specific, tissue-specific, temporary specific, or signal-inducible signals or agents external to signals or external agents.; these elements can be located in the 5 'or 3' regions of the original intron sequence. By "operably linked" it is meant that a gene and one or more regulatory sequences are connected in a manner that allows expression of the gene when suitable molecules (e.g., transcriptional activating proteins) are linked to the regulatory sequences. By "FREAC3 binding site" is meant linking a DNA sequence that allows the specific binding of FREAC3 to the DNA sequence. A FREAC3 binding site is the sequence aGTAAA (T / c) AAAca (SEQ ID NOs: 3 and 4). By "detectably labeled" is meant any means for labeling or identifying the presence of a molecule, for example, an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P, 33P or 35S) and non-radioactive labeling (e.g., chemiluminescent labeling, e.g. labeling by fluoroscein). By "purified antibody" is meant an antibody that is at least 60 percent, by weight, without naturally occurring organic proteins and molecules with which it is naturally associated. Preferably, the preparation is at least 75 percent, more preferably 90 percent, and still more preferably at least 99 percent, by weight, antibody, for example, an antibody specific for mutant FREAC3. A purified antibody can be obtained, for example, by affinity chromatography using recombinantly produced protein or conserved motif peptides and standard techniques. By "specifically binding" is meant an antibody that recognizes and binds to a FREAC3 polypeptide but does not bind to unrelated polypeptides. A preferred antibody specifically binds to a mutant FREAC3 mutant or polypeptide but does not substantially recognize or bind FREAC3 wild type molecules in a sample, eg, a biological sample, which naturally includes protein. A preferred antibody binds to a FREAC3 polypeptide having a mutation or polymorphism at one or more of the positions indicated in Figure 2. "Nucleotide coding" means a nucleotide with the coding region of FREAC3. For example, the first residue of the initiating methionine codon of FREAC3 is coding nucleotide 1, and the first residue of the second codon in FREAC3 is coding nucleotide 4. The numbering of all coding nucleotides is relative to the nucleotide coding 1. Brief Description of the Drawings Figure 1 is a diagram showing the genetic mapping of the gene or the IRID1 genes. Figure 2 is a diagram showing the cDNA and the amino acid sequence of FREAC3. Figure 3 is a diagram showing radiographs showing mutated FREAC3 gene sequences. "Figure 4 is a diagram showing Northern blot analysis of FREAC3 expression in human tissues, Figure 5 (ad) is a diagram showing expression studies of the FREAC3 Mfl mouse homolog in developing the murrine eye. of the Preferred Modalities The genetic link, the genome mismatch scan, and the analysis of patients with chromosomal alterations of chromosome 6 have indicated that an important place for the development of the anterior segment of the eye and for the development of glaucoma, IRID1 is located at 6p25 FREAC3, a member of the hairpin / winged helix transcription factor family, has also been mapped in 6p25, FREAC3 DNA sequencing in five IRID1 families and 16 sporadic patients with DNA defects. Previous segment revealed three mutations: a predicted 10 base pair deletion that causes a phase shift and a premature truncation of protein before the FREAC3 hairpin DNA binding domain and two nonsense amino acid mutations conserved within the FREAC3 hairpin domain. These nonsense mutations could impair DNA binding and the nuclear localization of the FREAC3 protein. A finding of FREAC3 mutations in three patients with ocular defects indicates that FREAC3 is involved in anterior segment dysgenesis and in glaucoma. Although numerous genes of the human hairpin family have been described, ours is the first to show that a mutation in a hairpin gene is below a human development disorder. Mfl, the homologous murine of FREAC3, is expressed in the developing brain, the skeletal system and the eye, consistent with FREAC3 that has a role in ocular development. The three putative inactivating mutations of FREAC3 and the pattern of Mfl expression in the developing eye are consistent with the FREAC3 haplo-insufficiency that underlies autosomal dominant glaucoma and anterior segment dysgenesis in IRID1 patients. The three mutations found in the FREAC3 gene presented in patients (one family, and two sporadic cases) were originally diagnosed with the Axenfeld-Rieger anomaly (ARA) form of IRID1. Mutational analysis, however, excluded FREAC3 from being below the disorders of the anterior segment in four other families with glaucoma and anterior segment dysgenesis linked with 6p25, and genetic linkage analysis actually excluded the FREAC3 gene in two of these families (Figure 1) . Interestingly, these four families (families IRID1 1, 2, 4 and 5) were originally diagnosed with IGDA, IGDA, FGI, and familial glaucoma with gonodisgenesis, respectively.

The FREAC3 mutations described above were found in patients with ARA. While all autosomal dominant IRID1 disorders include glaucoma, iris hypoplasia, and anterior angle defects, ARA patients additionally have a previously displaced, prominent Schwalbe line, attached to peripheral iris strips that bypass the iridocorneal angle, and displaced pupils. , characteristics not typically seen in GIDA, FGI, or goniodisgénesis. The four remaining IRID1 families could then be phenotypically as well as genetically distinct from the ARA family and the patients found to carry FREAC3 mutations. Our findings demonstrate that although FREAC3 mutations result in anterior segment defects and glaucoma in some patients, at least one place more involved in the regulation of eye development must also be located in 6p25. Knowledge of the gene defect in IRID1 families will allow the immediate monitoring and presymptomatic treatment of glaucoma that IRID1 patients frequently develop. Moreover, mutations of the IRID1 gene may be responsible for a significant portion of glaucoma patients, not clinically diagnosed with IRID1, since not all IRID1 patients, even within IRID1 families, have the iris defects used to diagnose IRID1. As the glaucoma developed by IRID1 patients is often difficult to treat with existing drugs, the detection of a FREAC3 mutation in a patient could indicate that the patient's glaucoma should be treated surgically. In the future, the detection of FREAC3 mutations in glaucoma patients could allow the separation of patients in two different subgroups of glaucoma, not only allowing the improved prediction of the patient's response to different glaucoma treatments, but also the design of better treatments for glaucoma. In this way, characterization of the FREAC3 gene will not only greatly increase our understanding of the development of the anterior segment, but also of the pathogenesis of glaucoma. Detection of FREAC3 mutations and altered expression levels FREAC3 polypeptides and nucleic acid sequences are of diagnostic use to identify patients who have an increased likelihood of having anterior segment dysgenesis and / or developing glaucoma. Mutations in FREAC3 that decrease FREAC3 expression or biological activity can be correlated with anterior segment defects and glaucoma in humans. A biological sample obtained from a patient can be analyzed to determine one or more mutations in the FREAC3 nucleic acid sequence using a mismatch detection approach (these mutations can also be detected in prenatal choices). Generally, these techniques involve the amplification of the polymerase chain reaction of FREAC3 genomic DNA or the PCR polymerase chain reaction amplification of FREAC3 mRNA from a patient sample, followed by identification of the mutation (i.e. lack of • coincidence) by either altered hybridization, aberrant electrophoretic gel migration, linkage or dissociation mediated by linkage proteins with mismatch, or direct nucleic acid sequencing. Any of these techniques can be used to facilitate the detection of mutant FREAC3, and each is well known in the art; the examples of particular techniques • res are described, without limitation, in Orita and collaborators. (Proc. Nati, Acad. Sci. USA 86: 2766-2770, 1989) and Sheffield et al. (Proc. Nati, Acad. Sci. USA 86: 232-236, 1989). Mismatch detection assays provide an opportunity to diagnose a FREAC3-mediated predisposition for glaucoma prior to the onset of symptoms. For example, a patient heterozygous for a FREAC3 mutation that decreases the biological activity of FREAC3 or the expression may not show clinical symptoms and still have a higher than normal likelihood of developing glaucoma. Even more, 0 certain alleles of the natural type of FREAC3 present in the population may increase the risk of developing other diseases of the eye. Given this diagnosis, a patient can take precautions to minimize their exposure to adverse environmental factors (for example, exposure to ultraviolet light5), to carefully monitor their medical condition (for example, through frequent physical examinations) and to take preventive measures. additional, such as using prophylactic medication or undergoing surgery or other preventive treatment. A decrease in the level of FREAC3 production can also provide an indication of a harmful or potentially harmful condition in a patient. FREAC3 expression levels can be tested by any standard technique. For example, FREAC3 transcriptional regulatory sequences can be analyzed for mutations that alter expression levels as a means to determine whether altered expression is likely, or FREAC3 transcription can quantify normal cells (e.g. peripheral blood) and these levels can be compared to FREAC3 transcription levels in the peripheral blood leukocytes of the test subject. The expression of FREAC3 in a biological sample (eg, a biopsy) can be monitored by standard Northern staining analysis or by polymerase chain reaction (see, for example, F., Ausubel et al., Current Protocols in Molecular Biology, John Wiley &; Sons, New York, NY, 1998; PCR Technology, Principles and Applications for DNA Amplification, H.A. Ehrlich, Ed., Stockton Press, NY; Yap and collaborators, Nucí. Acids Res. 19: 4294, 1991). The FREAC3 expression assays described above can be carried out using any biological sample (eg, any biopsy sample, blood sample, or other cells or tissue samples) in which FREAC3 is normally expressed. The identification of a mutated FREAC3 gene can also be tested using these sources for the samples used. Low levels of FREAC3 expression, or a mutation in the FREAC3 gene, identifies a patient with an increased risk of anterior segment dysgenesis and glaucoma. Alternatively, a FREAC3 mutation, particularly as part of a diagnosis of degenerative disease disposition associated with FREAC3, can be tested using a sample of AD? from any cell, for example, by means of mismatch detection technique. Preferably, the sample of AD? it is subjected to polymerase chain reaction amplification before analysis. In yet another approach, immunoassays are used to detect or monitor the expression of the FREAC3 protein in a biological sample. Polyclonal or monoclonal antibodies specific for FREAC3 (produced by standard techniques) can be used in any standard immunoassay format (eg, ELISA, Western blotting, or RIA) to measure FREAC3 polypeptide levels. These levels would be compared with FREAC3 levels of the wild type. For example, a decrease in the production of FREAC3 may indicate an increased risk of developing glaucoma. Examples of immunoassays are described, for example, in F., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N, 1998. Immunohistochemical techniques can also be used for the detection of FREAC3. For example, you can get a • sample of a patient's tissue, section, and stain for the presence of FREAC3 using an anti-FREAC3 antibody and any standard detection system (eg, one that includes a secondary antibody conjugated with horseradish peroxidase). General guidance regarding this technique can be found in, for example, Bancroft and Stevens (Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and F., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1994. Assays for the identification of crue compounds modulate or mimic the biological activity of FREAC3. Methods to observe changes in the biological activity of FREAC3 are exploited in high-throughput assays for the purpose of identifying compounds that modulate mutant FREAC3 transcription activity or natural type. Compounds that mimic the activity of FREAC3 can also be identified by these assays. In addition, the compounds that modulate the FREAC3 gene transcript itself can be identified; in some cases, it may be desirable to increase or decrease FREAC3 protein levels (eg, decrease FREAC3 mutant levels or increase wild-type levels). These identified compounds may have utility as therapeutic agents in the treatment or prevention of glaucoma or anterior segment dysgenesis. Test compounds • In general, novel drugs for the prevention or treatment of anterior segment dysgenesis or glaucoma that work modulating or mimicking the biological activity of FREAC3 are identified from large libraries of either extracts of the natural product or synthetic (or semi-synthetic) extracts ) or chemical libraries according to methods known in the art. Experts in the field of drug discovery and development will understand that the precise source of the test extracts or compounds is not critical to the method (s) of analysis of the invention. In accordance with the above, virtually any number of extracts of chemical products or compounds can be analyzed using the exemplary methods described herein. Examples of these extracts or compounds include, but are not limited to, extracts based on plants, fungi, prokaryotes or animals, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available to generate random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, compounds based on saccharides, lipids, peptide, nucleic acid. Synthetic compound libraries are commercially available from Brandon Associates (Merri ack, NH) and Aldrich Chemical (Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from various sources, including Biotics (Sussex, UK), Xenova (Slogh, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, USA (Cambridge, MA). In addition, libraries are produced naturally and synthetically, if desired, according to methods known in the art, for example, by standard extraction and fractionation methods. In addition, if desired, any library or compound is easily modified using standard chemical, physical, or biochemical methods. In addition, those skilled in the art of drug discovery and development readily understand that methods for unreplication (eg, taxonomic unreplication, biological re-application, and chemical re-replication, or any combination thereof) or the elimination of replicates or • repetitions of materials already known for their therapeutic value to treat or prevent glaucoma or segment dysgenesis above will understand being employed whenever possible. When a crude extract is found to modulate or mimic the biological activity of FREAC3, further fractionation of the positive forward extract is necessary to isolate the chemical constituents responsible for the effect observed. In this way, the goal of the extraction, fractionation, and purification process is the characterization and careful identification of a chemical entity within the crude extract that has an activity that avoids or improves the disorder of the anterior segment or glaucoma, via the modulation or mimicry of the biological activity of FREAC3 or the expression. The same assay described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test the derivatives thereof. The methods of fractionation and purification of these heterogeneous extracts are known in the art. If desired, the compounds shown to be useful agents for the treatment are chemically modified according to methods known in the art. The compounds identified as being of therapeutic value can subsequently be analyzed using a standard animal model for anterior segment dysgenesis or glaucoma. One of these models is a mouse that has one or both Mfl genes (murine FREAC3) cut out or mutated in the positions corresponding to those described herein for FREAC3 (see Figure 2). Another of these models is a mouse (either with genes of the wild type, or with genes cut out or imitated Mfl) that contains a mutated human FREAC3 transgene. Analysis for compounds that modulate FREAC3 mRNA or protein expression FREAC3 cDNA can be used to facilitate the identification of compounds that increase or decrease the expression of the FREAC3 protein. In one approach, candidate compounds, in various concentrations, are added to the culture medium of cells expressing FREAC3 mRNA. The expression of FREAC3 mRNA is then measured, for example, by Northern blot analysis (F., Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, NY, 1994) using DNA, cDNA, or fragments of FREAC3 RNA as a hybridization probe. The level of FREAC3 mRNA expression in the presence of the candidate compound is compared to the level of FREAC3 mRNA expression in the absence of the candidate compound, all other factors (e.g., cell type and culture conditions) are same. Cells that normally express FREAC3, such as those derived from skeletal muscle, heart, liver, kidneys, pancreas, prostate, testes, ovaries, fetal kidney, and peripheral blood leukocytes can be used. Moreover, cells whose FREAC3 promoter is not normally active can be provided with a FREAC3 promoter exogenously derived fused with FREAC3 or with a reporter gene, for example, luciferase or β-galactosidase (see below), and used in the described assays at the moment. As an alternative approach to detect compounds that regulate FREAC3 at the level of transcription, candidate compounds can be tested for the ability to regulate the expression of a reporter gene whose expression is driven by a FREAC3 gene promoter. For example, a cell that normally expresses FREAC3, such as a cell derived from skeletal muscle, or alternatively, a cell that does not normally express FREAC3, such as a cell derived from the colon, can be transfected with an expression plasmid that includes a gene. luciferase (or another reporter) operably linked to the FREAC3 promoter. The candidate compounds can then be added, in various concentrations, to the culture medium of the cells. The luciferase expression levels can then be measured by subjecting the transfected cells of the treated compound to luciferase assays known in the art (such as the set of luciferase assay system used herein that is commercially available from Promega), and quickly assessing the level of luciferase activity in a luminometer. The level of luciferase expression in the presence of the candidate compound is compared to the level of luciferase expression in the absence of the candidate compound, all other factors (e.g., cell type and culture conditions) are left the same. An increase in luciferase expression indicates a compound that increases FREAC3 gene expression; conversely, a decrease in luciferase expression indicates a compound that decreases the expression of the FREAC3 gene. The effect of candidate compounds on FREAC3-mediated gene expression may instead be measured at the translation level using the general approach described above with standard protein detection techniques, such as Western blotting or immunoprecipitation with a antibody specific for FREAC3 (for example, the antibody specific for FREAC3 described herein). Analysis of compounds that modulate or mimic the biological activity of FREAC3 The compounds can also be analyzed for the ability to modulate the biological activity of mutant FREAC3 or the wild-type, eg, transcription activation of a target gene by FREAC3. In this approach, the level of transcription mediated by FREAC3 in the presence of a test compound is compared to the level of transcription in the absence of the test compound, under equivalent experimental conditions. Again, the analysis may begin with a set of candidate compounds, from which one or more useful modulator compounds are isolated in a stepwise manner. The activation of transcription of a target gene by FREAC3 can be measured by any standard assay, for example, those described herein. Another method to detect compounds that modulate the biological activity of FREAC3 is to look for compounds that physically interact with a given FREAC3 polypeptide. These compounds are detected by adapting yeast two-hybrid expression systems known in the art. These systems, which detect protein interactions using a transcription activation assay, are generally described by Gyuris et al. (Cell 75: 791-803, 1993) and Field et al. (Nature 340: 245-246, 1989), are commercially available from Clontech (Palo Alto, CA). Below are examples of high production systems useful for evaluating the efficacy of a molecule or compound for treating or preventing anterior segment dysgenesis and / or glaucoma caused by a mutant FREAC3 protein, or whose course is affected by a FREAC3 protein of the type natural . Reporter gene assays for compounds that modulate or imitate the FREAC3 transcription activity The assays that used the reporter gene product detection are extremely sensitive and easily docile to automation, making them ideal for the design of high production searches. In the cloned DNA fragments that encode a transcription control region whose activity is regulated by FREAC3, they are easily inserted, by means of their DNA cloning, into a reporter gene factor, thereby placing a vector-encoded reporter gene under control of transcription of the transcription control region regulated by FREAC3. The transcription activity of a promoter operably linked to a reporter gene can be observed directly and quantified as a function of the reporter gene activity in a reporter gene assay. These plasmid vectors or viral reporter gene contain cassettes that encode reporter genes such as lacZ / β-galactosidase, green fluorescent protein, and luciferase, among others. Trials for gene activity • Reporter can use, for example, co-calorimetric, guimioluminescent, or fluorometric detection of the products of the reporter gene. At appropriate points in time, cells treated with test compounds are lysed and subjected to appropriate reporter assays, for example, a calorimetric or chemiluminescent enzymatic assay for lacZ / β-galactosidase activity, or fluorescent detection or of green fluorescent protein (GFP). Changes in reporter gene activity of samples treated with test compounds, relative to the reporter gene activity of appropriate control samples, indicate the presence of a compound that modulates FREAC3 transcription activity. In one embodiment, a gene construct activated by FREAC3 could include a reporter gene such as lacZ or green fluorescent protein (GFP), operably linked to a promoter from the gene that is transcriptionally activated by FREAC3. Alternatively, an artificial FREAC3 activated gene can be created by fusing multiple copies of an artificial FREAC3 linkage site that is known in the art (aGTAAA (T / c) AAAca; (SEQ ID NOs: 3 and 4)) upstream of the promoter minimal, such as the thymidine kinase promoter of herpes simplex. These regulatory sequences can be fused to a downstream reporter gene (e.g., lacZ), and an alteration modulated by the test compound in binding the FREAC3 to the FREAC3 linkage site will be observed as a level change in reporter gene activity . A FREAC3 activated gene construct can be present within the genomic DNA of a cell to be used to analyze a test compound, or it can be introduced transiently. A second gene construct, comprising a second reporter gene operably linked to a second promoter (such as an SV40 promoter), is included as an internal control. Therefore, the change in the activity of a reporter gene operably linked in the transcription control sequence that is an objective of FREAC3 reflects the ability of a test compound to modulate the transcription activity of FREAC3. FREAC3 can be expressed naturally within the test cell, such as a cell derived from skeletal muscle, heart, kidney, pancreas, prostate, testes, ovaries, peripheral blood leukocytes, or fetal kidney, or it can be artificially expressed to starting from a nucleic acid encoding FREAC3 introduced permanently or transiently; Nucleic acids that encode either wild-type or FREAC3 mutant forms can be used. Also, reporter gene assays can be performed on cells that lack FREAC3, in order to isolate molecules that mimic FREAC3 activity. In order to identify compounds that increase or decrease the transcription of the FREAC3 gene itself, constructs of the reporter gene that employs the FREAC3 promoter region can be used. Linked enzyme-linked immunosorbent assays for compounds that modulate or mimic the transcription activity of FREAC3 Enzyme-linked immunosorbent assays (ELISA) are easily incorporated into high-throughput assays designed to test a large number of compounds for their ability to modulate activity biological of a given protein. When used in methods of the invention, changes in the level of a given reporter protein (eg, the product of a gene that is transcriptionally activated by FREAC3), relative to a control, reflect compounds that modulate FREAC3 biological activity ( or that mimics the activity of FREAC3, depending on the tests). The presence of FREAC3 polypeptide can also be monitored in order to test compounds that influence the transcription, translation, or stability of FREAC3 mRNA or polypeptide. The test samples may be cells, cell lysates, or purified or partially purified molecules. The cells can be derived from the heart, esguelético muscle, kidney, pancreas, prostate, testes, ovaries, fetal kidney, or leukocytes of peripheral blood, or they can be other types of cells that are genetically designed technically to express FREAC3 via a gene that encodes FREAC3 introduced permanently or transitorily. Protocols for ELISA can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & amp;; Sons, New York, NY, 1998. In one embodiment, the so-called "sandwich" ELISA, treated samples comprising used cells or purified molecules are loaded into wells of microtitre plates coated with "capture" antibodies. The unbound antigen is washed, and a second antibody, coupled with an agent to allow detection, is added. Agents that allow detection include alkaline phosphatase (which can be detected after the addition of colorimetric substrates such as p-nitrophenol phosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham) or compounds fluorescent, such as FITC (which can be detected by fluorescence polarization or time resolved fluorescence). The amount of antibody binding, and hence the level of reporter protein expressed by a gene that is transcriptionally activated by FREAC3, is easily quantified on a microtiter plate reader. For example, an increased level of an indicator protein in a treated sample, relative to the level of an indicator protein in an untreated sample, indicates a test compound that increases the transcription activity of FREAC3. It is understood that adequate controls for each assay are included as a baseline reference. High throughput assays for the purpose of identifying compounds that modulate or mimic the biological activity of FREAC3 can be performed using treated samples of cells, cell lysates, baculovirus lysates, and purified or partially purified molecules. Trap interaction assays Two hybrid and one hybrid methods, and modifications thereof, are used to select compounds that modulate the physical interactions of FREAC3 with other molecules (eg, proteins or nucleic acids). These assays can also be used to identify novel proteins that interact with FREAC3, and therefore can be naturally occurring regulators of FREAC3. These assays are well known to those skilled in the art, and can be found, for example, in F., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998. DNA binding assays The binding of FREAC3 mutant or wild-type to the binding site sequence in vi tro of FREAC3 (aGTAAA (T / c) AAAca; SEQ ID NOs: 3 and 4 ) can be used to select compounds that modulate the biological activity of FREAC3. One method by which these changes are quantified is by means of an ELISA type test. The samples containing FREAC3 are incubated with the test compounds as described above, plus an oligonucleotide encoding the FREAC3 binding site (such as aGTAAA (T / c) AAAca) that is fixed to a solid support (e.g. a filter, or a microtiter well). After allowing FREAC3 to interact with its cognate binding sequence and FREAC3 which did not bind was washed, the amount of FREAC3 binding to the immobilized nucleotide can be quantified by subsequent incubation with an ethylated antibody. A compound which increases or decreases the amount of mutant FREAC3 bound to the immobilized oligonucleotide indicates a compound which may be useful for the treatment or prevention of glaucoma or anterior segment dysgenesis. Secondary analysis of test compounds that appear to modulate or mimic the transcription activity of FREAC3. After the test compounds that appear to have FREAC3 modulation activity are identified, it may be necessary or desirable to subject these compounds to other tests. The invention provides these secondary confirmatory assays. For example, a compound that appears to modulate the biological activity of the mutant FREAC3 (ie, induces the mutant FREAC3 to have activity approaching wild-type FREAC3) in previous tests can be further tested to determine the effect of the compound on FREAC3. of the natural type.

In the last stages of the test, the live test is carried out to confirm that the compounds initially identified as affecting the activity of FREAC3 have the effect predicted in FREAC3 in vivo. In the first round of live tests, the compound is administered to animals either with wild type genes or FREAC3 mutants by one of the means described in the therapy section below. The tissue of the eye, or other tissues expressing FREAC3 (ie see Figure 2) is isolated in hours to days after treatment, and is subjected to tests as described above. Construction of transgenic animals and animals of cut-off genes Animals of FREAC3-cut genes, such as FREAC3 knockout mice, can be developed by homologous recombinations. Animals that overproduce FREAC3 mutant can be generated by integrating one or more FREAC3 sequences in the genome of these animals, according to standard transgenic techniques. A directed type of replacement type, which could be used to create a cut-off gene model, can be constructed using an isogenic genomic clone, for example, from a mouse strain such as 129 / Sv (Stratagene Inc., LaJolla, CA). The target vector can be introduced into embryonic stem (ES) cells by electroincorporation to generate embryonic stem cell lines that carry a deeply truncated form of a FREAC3 gene. To generate chimeric founder mice, the target cell lines are injected into an embryo in the mouse blastula stage, and the mice that transmit the FREAC3 shortened gene to their descendants are identified. Mice with heterozygous FREAC3 trimmed gene can be bred to be homozygous, so that no FREAC3 is expressed. Mice with a shortened gene provide the means, in vivo, to select therapeutic compounds that modulate anterior segment dysgenesis or the outbreak or progression of glaucoma via the FREAC3-dependent pathway or affected by FREAC3. Therapeutic use of compounds identified by high production systems A compound that promotes an alteration in the expression or biological activity of the FREAC3 protein is considered particularly useful in the invention; this molecule can be used, for example, as a therapy to increase cellular levels of biologically active FREAC3 and therefore exploit the role of FREAC3 polypeptide in anterior segment formation, differentiation of the trabecular network, and pressure regulation intraocular This could be advantageous in the prevention and / or treatment of anterior segment dysgenesis and / or glaucoma. Molecules are found, by the methods described above, that effectively modulate the expression of the FREAC3 gene or the activity of the polypeptide can be further tested in animal models described above. If they continue to function successfully in a live setting, they can be used as therapy to increase the biological activity of FREAC3 and / or expression, as appropriate. The compounds identified using any of the methods described herein, can be administered to patients or experimental animals with a pharmaceutically acceptable diluent, carrier, or excipient in unit dosage form, as described in the therapy section below. Therapy The therapeutic molecules are identified using any of the methods described herein, they can be administered to patients or experimental animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice can be employed to provide convenient formulations or compositions for administering these compositions to patients or experimental animals. Although intravenous administration is preferred, any suitable route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, the formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. The methods well known in the art for making • formulations are found, for example, in "Remington Pharmaceutical Sciences". Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biodegradable, biocompatible lactide polymer, copolymer • Lactide / glycolide, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for administration of the molecules of the invention include ethylene-vinyl acetate-5-copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The formulations for inhalation may contain excipients, for example, lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops , or as a gel. The following examples also illustrate the invention. This does not mean that they limit the invention in any way. Example 1; General methods 5 Clinical data The clinical presentations within the IRID1 1-5 families have all been previously reported. Families 1 and 2 were originally diagnosed with an iridogoniodis-genesis (IGDA) abnormality (Mears et al., Am. J. Hum. Genet., 59: 1321-7, 1996, Pearce et al., Can. J. Ophthalmol. 7-10, 1983), family 3 with Axenfeld-Rieger anomaly (Gould et al., Am. J. Hum. Genet, 61: 765-768, 1997), family 4 with familial iridogoniodysplasia (Jordan et al., Am. J. Hum Genet, 61, 1997) and family 5 with goniodisgenesis and glaucoma (Morissette et al., Am. J. Hum Genet, 61: A286, 1997). The five IRID1 families demonstrated phenotypic variability but affected individuals typically present with iris hypoplasia, iridocorneal angle defects (goniodisgenesis), and increased intraocular pressure with their subsequent risk of glaucoma. Affected individuals within family 3 additionally presented a previously displaced, prominent Schwalbe line (posterior embryotoxon) attached to the peripheral iris strips using the iridocorneal angle, and displaced pupils (corectopia). Some of the patient families also had histories of congenital heart defects. Sixteen unrelated individuals who presented anterior segment dysgenesis were also studied. The study and collection of blood samples from all individuals included in this report were approved by the Research Ethics Board of the University of Alberta Medical School. Fiery polvorum markers The novel polymorphisms were detected in exon 5 of the NAD (P) H gene: quinone oxidoreductase-2 (NQ02) by direct sequencing of amplified polymerase chain reaction products from the key recombinant branches of the families IRID1. The primers for exon 5: forward 5 '-gcttcattccgaatcaccag-3' (SEQ ID NO: 5), inverse 5'-gtcccctccctccaactatc-3 '(SEQ ID NO: 6). The primers were designed using primer 3, available from the Whitehead Institute for Biomedical Research (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3. Egi). The two polymorphisms within NQ02 exdn 5 both affect MspI sites at the positions of 111 base pairs and 188 base pairs of the polymerase chain reaction product of 250 base pairs, generating a polymorphic system of four alleles. Physical Mapping The preliminary physical mapping for the IRID1 6p25 region was obtained from the Whitehead Institute for Biomedical Research whose web site is (http: // www-genome, wi .mit. E-du /). The human bacterial artificial chromosome library (BAC) (Kim et al., Genomics 34: 213-218, 1996; Shizuya et al., Proc. Nati, Acad. Sci. USA 89: 8794-8797, 1992) was selected by polymerase chain with STSs / ESTs mapped to the region, according to the Research Genetics protocols. The selected clones were mapped by fluorescence on site hybridization (FISH) to confirm the cytogenetic localization, and then analyzed • to determine the STS content to determine the order and overlap between clones. Scanning of sequences The scan of sequences was performed in the BAC RMC06B016. This BAC, containing an insert of approximately 150 kb in size, was randomly sheared and fragments • variants of 2-3 kb were subcloned into the vector M13mpl8. Sequences were obtained from 509 subclones using ABL 373 and 377 automated sequencers and assembled in contiguous using Seqman (DNASTAR). The contiguous ones were investigated to determine the coding sequence using BLAST 2.0 against the GenBank and the dEST database. GRAIL 1.2 was used to predict the coding sequence not represented in the existing databases. • Detection of mutation The fragments were amplified from the FREAC3 0 gene of a single exon using primers designated by primer 3 (see Table 1). Dimethyl sulfoxide (final concentration of 5-10 percent) was added to the polymerase chain reactions to alleviate secondary structure problems created by the very high GC content of FREAC3. The products of the polymerase chain reaction were purified with QIAquick columns (QIAGEN, Los Angeles, CA) then sequenced directly via 33 P cycle sequencing (Amersham, Malvern, PA). Mutations were confirmed in affected individuals and investigated on 100 control chromosomes by the following methods: suppression of 10 base pairs (from nt91-100) was detected through the polymerase chain reaction products on 1.5 percent of agarose / 1.5 percent NuSieve of electrophoretic gels. The G245C mutation was detected through the loss of an Alu I site. The C261G mutation was detected through the generation of the Bsp Hl site. The insertion of polymorphisms (GGC375ins and GGc347ins) were detected by sequencing both patients and control individuals. Expression Analysis Expression of FREAC3 was determined by Northern blot analysis of commercially available filters (Clontech, Palo Alto, CA) containing poly (A) + RNA selected from a variety of adult and fetal tissues. To avoid cross-hybridization with other hairpin-related genes, the probe for FREAC3 was selected from the 3 'region (nucleotides 1192-1690, see Figure 2). Hybridization and washing were performed according to the manufacturer's protocols. The human β-actin control probe, provided by the manufacturers, was used to equalize the load differences. Mfl is the mouse homolog of the FREAC3 gene. A mouse with Mfl trimmed gene was created by homologous recombination in embryonic stem cells in which the corresponding amino acid sequences 50-553 and the 3 'untranslated region ^ = W of the Mfl gene were replaced by a lacZ / PGKneor cassette in the frame with the first AUG. The expression of the MflLacZ gene was detected by X-Gal spotting of the eye sections from mouse embryos MflLacZ / + (+/-) and MfILacZ / MflLacZ (- / -) (14.5 dpc). Example 2: Genetic refinement of the location of the IRID1 site Genetic linkage analysis was used to refine the location of the IRID1 site. Figure 1 shows a schematic diagram of chromosome 6, illustrating the genetic mapping of the IRIDl or. The cumulative genetic distances (in cM) of the telomere are indicated on the left. Haplotypes of the cosegregant disease in the five IRID1 families are represented by filled rectangles. The key individuals are identified in the upper part of the Figure, with the disease status indicated below. Location of the site associated with anterior segment dysgenesis and glaucoma in families 1 and 4 are indicated on the right. The results of the known and novel 6p25 polymorphic marker analyzes in five IRID1 families were generally consistent with the location of IRID1 between D6S1600 and polymorphisms in the NAD (P) H gene: quinone oxidoreductase (NQ02) (Figure 1). However, an unaffected individual (IRID1 family 1; VIII: 1) had an apparent growth event placing IRID1 distal to D6S344 (Figure 1). This observation is not consistent with FREAC3 being a candidate gene for IRID1 in this family (see below). IRID1 • it is thought to be a fully penetrating autosomal dominant disorder. However, the non-penetration of IRID1 in the individual VIII: 1 could not formally be considered as a possible explanation of this apparent mapping discrepancy. Example 3: FREAC3, a candidate gene located in the critical region of IRID1 • In order to physically clone the IRID1 interval, twenty-nine BACs were obtained by selecting the BAC genomic library with linked sites of known sequences (STSs) and ethiguetas of expressed sequences ( ESTs). It was found that GAC RMC06B016 contained the distal flange marker D6S344 and was positive with the primers designed from the published partial sequence of FREAC3, a gene previously mapped to 6P25 (Larsson et al., Genomics 30: 464-469, • ninn ninety five) . FREAC3 is a member of the gene seed of hairpin transcription factor shown as involved in the development, the specific development of the cell, and oncogenesis. FREAC3 was reported to be localized within 20 kb of the 6p25 translocation cut-off point in the individual with an unbalanced karyotype (t (2.6) (q35, p25)) presented with a variety of clinical findings including glaucoma (Nishimura and collaborated ¬ res, Am. J. Hum. Genet 61: A21, 1997).

The approximately 80 percent DNA sequence of BAC RMC06B016, or approximately 120 kb of sequence, was dmined as a rapid means of characterizing FREAC3 and • identify additional genes within the critical region IRID1. The hairpin-like region BAC RMC06B016 was identical to the partial DNA sequence of the FREAC3 gene. Additional sequence analysis revealed that FREAC3 has a less clear open intron of 1659 base pairs (SEQ ID NO: 1) and is predicted to quantify a protein (SEQ ID NO: 2) of 553 • amino acids (Figure 2). Mfl, the murrain gene homologue to FREAC3, is also predicted to encode a protein of 553 amino acids in length and has been mapped to mouse chromosome 13 in a region of synteny preserved with human 6p25 (database, MG 5 Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine, 1998, URL: http://www.informatics.jax.org/). The human FREAC3 gene and the mouse Mfl gene share 89 percent of their nucleotide sequence through the coding region with the highest degree of identity (96 percent) seen over 0 the 330 nucleotides of the domains of the head of fork. Above all, the identity of the protein level was found to be 92 percent with 100 percent identity from the hairpin DNA binding region. Example 4: FREAC3 mutations in patients with anterior segment 5 and glaucoma dysgenesis The FREAC3 gene was investigated for mutations by direct DNA sequencing of polymerase chain reaction products from affected individuals from the five IRID1 families linked to 6p25 polymorphic sites and in 16 additional unrelated individuals with anterior segment dysgenesis. Five nucleotide alterations of FREAC3 were found. Figure 2 shows the nucleotide and the predicted amino acid sequence of FREAC3. The open reading frame is 1659 base pairs in length, predicted to encode a protein of 553 amino acids. The hairpin domain, which encompasses amino acids 69-178, is framed. The arrowheads indicate the two locations of the polymorphic GGC insertions (see below). Three FREAC3 mutations dted in patients with anterior segment dysgenesis and glaucoma are indicated by 1, 2 and 3 (Figure 2); Enzyme horizontal bars of the nucleotide sequence indicating the affected nucleotides. A suppression of 10 base pairs of base pairs 93-102, which is 5 'of the region encoding the hairpin domain of FREAC3, was found in an individual with Axenfeld-Rieger anomaly (ARA) and glaucoma ( Patient # 1, Figure 3). This alteration occurs after the initiation codon, and is predicted to result in a mutation of frame shift and premature arrest after 10 amino acids. A second alteration, a transversion from G to C at the position of nucleotide 245 results in a Ser82Thr mutation in helix 1 of the FREAC3 hairpin domain, was identified in family 3 IRID1 (originally diagnosed with ARA, Figure 3). The G245C mutation abolished an Alu I restriction enzyme site and was found to segregate with the phenotype of anterior segment dysgenesis / glaucoma in all affected members in family 3. This amino acid position is invariably a serine in more than 80 yeasthead family genes from yeast to human. The distantly related QRF1 gene (factor I rich in glutamine Q) has a threonine from a serine residue at this position within helix 1. However, since the QRF1 DNA binding domain is only 84 amino acids in length in comparison With 110 amino acids for hairpin genes, QRF1 could fall out of the hairpin gene family. Consistent with this notion, QRF1 appears to bind to DNA differently than predicted for hairpin-head protein, and therefore may not require a serine in this position, unlike all other hairpin genes. Site-directed mutagenesis of serine and two flanking tyrosines in the hairpin gene related HNF-3? It clears the DNA binding activity. The third mutation, a transversion from C to G at the position of nucleotide 261 that would result in a nonsense mutation Ile87Met in helix 1 of the FREAC3 hairpin domain (Figure 2), was identified in an individual diagnosed with ARA and glaucoma (patient # 2, Figure 3). This C261G mutation creates a Bsp Hl restriction enzyme site. This position within helix 1 is an isolucine in more than 88 percent of the hairpin genes, and had never been reported as methionine. Interestingly, also as with the putative DNA binding domain of FREAC3, both the Ser82Thr mutation and the Ile87Met nonsense are presented with a conserved region shown that acts as a necessary and sufficient nuclear localization signal for the nuclear direction of the gene of related hairpin family, HNF-3ß. These three FREAC3 nucleotide alterations were not observed in more than 100 uninfected chromosomes from normal controls. Figure 3 shows autoradiographs of sequence analysis of the mutations identified in the IRID1 3 family and in two patients with anterior segment dysgenesis and glaucoma. The polymerase chain reaction products were amplified from DNA samples in the patient and sequenced directly. The normal sequences are shown on the left, the sequences of the affected individuals are shown on the right. The sequence of reverse primer is shown in each case with the lanes representing GATC bases from left to right. The positions of the mutations are shown to the right and the predicted effects of the FREAC3 DNA mutations are indicated more to the right. Two alterations, GGC375ins and GGC447ins, each involving the insertion of an extra triplet GGC in two separate GGC repeats within the FREAC3 coding region (Figure 2) were found in both the patients and in the control individuals. These alterations are therefore presumed to be polymorphisms not associated with IRID1 of FREAC3. Example 5: FREAC3 expression studies Figure 4 shows a Northern blot analysis for the expression of FREAC3 in human, adult and fetal tissues. The filters were hybridized with a FREAC3 probe (upper panels), and a β-actin control probe (lower panels). A 4.4 kb FREAC3 mRNA transcript was detected by Northern blot analysis and found to be widely expressed in adult and human fetal tissues. The highest expression of FREAC3 was observed in the kidney, heart and peripheral blood leukocytes of adults, and in the fetal kidney (Figure 4). An alternative transcript of 4.0 kb size was also detected in the fetal kidney, possibly suggesting an alternative promoter or polyadenylation site that is being used in this tissue. Polymerase chain reaction analyzes indicated that FREAC3 is also expressed in human fetal cranial facial RNA and in the adult iris. Figure 5 (a-d) shows the expression pattern of the homozygous and heterozygous MflLacZ embryos in MflLacz. The expression of Mfl in MflLacZ / + (+/-) and the MflLacZ / MflLacZ (- / -) embryos is indicated by the spotted lacZ observed in photographs of sections of the developing eye in 14.5 dpc mice. The framed regions of panels 5a and 5b, respectively, are shown amplified in panels 5c and 5d. He • blue stained tissue indicates lacZ expression regions, which corresponds to the Mfl expression abundant in the periocular mesenchyme that develops the eyelids and the anterior segment. In the developing eye, lacZ staining was abundant in the periocular mesenchyme, in the developing eyelids and in the anterior segment (Figure 5 (a-d)). The LacZ activity was also observed in the mesenchyme of the hind quarter, heart, and the perichondrium of the ribs. The expression pattern of the murine homologue of the FREAC3 gene and the fact that the mice with homozygous Mfl cut-off gene develop severe eye anomalies and hydrocephalus, are very consistent with the hypothesis that FREAC3 has a role in the development of the eye. The relatively less severe abnormalities observed in IRID1 patients compared to mice trimmed in Mfl homozygotes that are simply a result of the fact that IRID1 patients are heterozygous and thus have a unique functional copy of the FREAC3 gene. Example £: IRID1 is genetically heterogeneous The complete DNA sequencing of the coding region of the FREAC3 gene in affected individuals in families 1, 2, 4, and 5 surprisingly failed to identify any mutation associated with IRID1 of FREAC3. In addition, the polymorphism analysis of GGC347ins in family 4 IRID1 genetically excluded the FREAC3 gene from underlying IRID1 in this family (Figure 1). The recombination event in VIII: 24 of the • IRID1 family 4 together with the recombination event within the unaffected individual VIII: 1 in the IRID1 1 family discussed above are consistent with the location of the second IRID1 between D6S1600 and D6S344 (Figure 1). Other modalities All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application were specifically and individually indicated as being incorporated by reference. Although the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of other modifications and this application is intended to cover any variation, use or adaptation of the invention which generally follows the principles of the invention and which includes the separations of the present disclosure are within known or customary practice within the art to which the invention pertains and may be applied to the essential features presented hereinabove, and remain within the scope of the appended claims.

LIST OF SEQUENCES < 210 > 1 < 211 > 1662 < 212 > DNA < 213 > Homo sap: Lens < 400 > 1 atgcaggcgc gctactccgt gtccagcccc aactccctgg gagtggtgcc ctacctcggc 60 ggcgagcaga gctactaccg cgcggcggcc gcggcggccg ggggcggcta caccgccatg 120 tgagcgtgta ccggccccca ctcgcaccct gcgcacgccg agcagtaccc gggcggcatg 180 gcccgcgcct acgggcccta ccgcagccca cacgccgcag aggacatggt gaagccgccc 240 tatagctaca tcgcgctcat caccatggcc atccagaacg ccccggacaa gaagatcacc 300 ctgaacggca tctaccagtt catcatggac cgcttcccct tctaccggga caacaagcag 360 ggctggcaga acagcatccg ccacaacctc tcgctcaacg agtgcttcgt caaggtgccg 420 cgcgacgaca agaagccggg caagggcagc tactggacgc tggacccgga ctcctacaac 480 atgttcgaga acggcagctt cctgcggcgg cggcggcgct tcaagaagaa ggacgcgttg 540 aaggacaagg aggagaagga caggctgcac ctcaaggagc cgcccccgcc cggcgccagc 600 ccccgcccgg cgccgccgga gcaggccgac ggcaacgcgc ccggtccgca gccgccgccc 660 gtgcgcatcc aggacatcaa gaccgagaac ggtacgtgcc cctcgccgcc ccagcccctg 720 tccccggccg ccgccttggg cagcggcagc gccgccgcgg tgcccaagat cgagagcccc 780 gacagcagca gcagcagcct gtccagcggg agcagccccc cgggcagcct gccgtcggcg 840 cggccgctca gcctggac gg tgcggattcc gcgccgccgc cgcccgcgcc ctccgccccg 900 ccgccgcacc atagccaggg cttcagcgtg gacaacatca tgacgtcgct gcgggggtcg 960 ccgcagagcg cggccgcgga gctcagctcc ggccttctgg cctcggcggc cgcgtcctcg 1020 cgcgcgggga J; cgcaccccc gctggcgctc ggcgcctact cgcccggcca gagctccctc 1080 tacagctccc cctgcagcca gacctccagc gcgggcagct cgggcggcgg cggcggcggc 1140 gcgggggccg cggggggcgc gggcggcgcc gggacctacc actgcaacct gcaagccatg 1200 agcctgtacg cggccggcga gcgcgggggc cacttgcagg gcgcgcccgg gggcgcgggc 1260 ggctcggccg tggacgaccc cctgcccgac tactctctgc ctccggtcac cagcagcagc 1320 tcgtcgtccc tgagtcacgg cggcggcggc ggcggcggcg ggggaggcca ggaggccggc 1380 cggcccacca caccaccctg aggccgcctc acctcgtggt acctgaacca ggcgggcgga 1440 gacctgggcc acttggcgag cgcggcggcg gcggcggcgg ccgcaggcta cccgggccag 1500 cagcagaact tccactcggt gcgggagatg ttcgagtcac agaggatcgg cttgaacaac 1560 tctccagtga acgggaatag tagctgtcaa atggccttcc cttccagcca gtctctgtac 1620 cgcacgtccg gagctttcgt ctacgactgt agcaagtttt ga 1662 < 21C) > 2 < 213. > 553 < 212í > PRT < 212? > Homo sapiens < 400 > 2 Met Gln Wing Arg Tyr Ser Val Being Ser Pro Asn Being Leu Gly Val Val 1 5 10 15 Pro Tyr Leu Gly Glu Gln Being Tyr Tyr Arg Wing Wing Wing Wing Wing 20 25 30 Wing Gly Gly Gly Tyr Thr Wing Wing Pro Wing Pro Met Ser Val Tyr Ser 40 45 His Pro Ala His Ala Glu Gln Tyr Pro Gly Gly Met Ala Arg Ala Tyr 50 55 60 Gly Pro Tyr Thr Pro Gln Pro Gln Pro Lys Asp Met Val Lys Pro Pro 65 70 75 80 Tyr Ser Tyr He Wing Leu He Thr Met Wing He Gln Asn Wing Pro Asp 85 90 95 Lys Lys He Thr Leu Asn Gly He Tyr Gln Phe He Met Asp Arg Phe 100 105 110 Pro Phe Tyr Arg Asp 'Asn Lys Gln Gly Trp Gln Asn Ser He Arg His 115 120 125 Asn Leu Ser Leu Asn Glu Cys Phe Val Lys Val Pro Arg Asp Asp Lys 130 135 140 Lys Pro Gly Lys Gly Ser Tyr Trp Thr Leu Asp Pro Asp Ser Tyr Asn 145 150 155 160 Met Phe Glu Asn Gly Ser Phe Leu Arg Arg Arg Arg Arg Phe Lys Lys 165 170 175 Lys Asp Wing Leu Lys Asp Lys Glu Glu Lys Asp Arg Leu His Leu Lys 180 185 190 Glu Pro Pro Pro Gly Wing Pro Pro Arg Pro Wing Pro Pro Glu Gln 195 200 205 Wing Asp Gly Asn Wing Pro Gly Pro Gln Pro Pro Pro Val Arg He Gln 210 215 220 Asp He Lys Thr Glu Asn Gly Thr Cys Pro Ser Pro Pro Gln Pro Leu 225 230 235 240 Ser Pro Ala Ala Ala Leu Gly Ser Gly Ser Ala Ala Ala Val Pro Lys 245 250 255 He Glu Ser Pro Asp Ser Ser Ser Ser Leu Ser Ser Gly Ser Ser 260 265 270 Pro Pro Gly Ser Leu Pro Ser Ala Arg Pro Leu Ser Leu Asp Gly Ala 275 280 285 Asp Ser Ala Pro Pro Pro Pro Ala Pro Ser Ala Pro Pro Pro His His 290 295 300 Ser Gln Gly Phe Ser Val Asp Asn He Met Met Thr Ser Leu Arg Gly Ser 305 -. 305 - 310 315 320 Pro Gln Ser Ala Ala Ala Glu Leu Ser Ser Gly Leu Leu Ala Ser Ala 325 330 335 Wing Wing Being Arg Wing Wing Gly Wing Pro Pro Leu Wing Leu Gly Wing 340 345 350 Tyr Being Pro Gly Gln Being Being Leu Tyr Being Being Pro Cys Being Gln Thr 355 360 365 Being Being Wing Gly Being Ser Gly Gly Gly Gly Gly Gly Ala Gly Ala Ala 370 375 380 Gly Gly Wing Gly Gly Wing Gly Thr Tyr His Cys Asn Leu Gln Wing Met 385 390 395 400 Ser Leu Tyr Ala Ala Gly Glu Arg Gly Gly His Leu Gln Gly Ala Pro 405 410 415 Gly Gly Wing Gly Gly Ser Wing Val Asp Asp Pro Leu Pro Asp Tyr Ser 420 425 430 Leu Pro Pro Val Thr Ser Be Ser Ser Ser Leu Ser His Gly Gly 435 440 445 Gly Gly Gly Gly Gly Gly Gly Gln Glu Gly Wing His His Pro Wing 450 455 460 Wing His Gln Gly Arg Leu Thr Ser Trp Tyr Leu Asn Gln Wing Gly Gly 465 470 475 480 Asp Leu Gly His Leu Wing Wing Wing Wing Wing Wing Wing Wing Wing Wing Gly 485 490 495 Tyr Pro Gly Gln Gln Gln Asn Phe His Ser Val Arg Glu Met Phe Glu 10 500 505 510 Ser Gln Arg He Gly Leu Asn Asn Ser Pro Val Asn Gly Asn Ser Ser 515 520 525 Cys Gln Met Wing Phe Pro Ser Ser Gln Ser Leu Tyr Arg Thr Ser Gly 530 535 540 15 Wing Phe Val Tyr Asp Cys Ser Lys Phe 545 550 < 210 > 3 - # < 211 > 12 20 < 212 > DNA < 213 > Homo sapiens < 400 > 3 agtaaataaa ca 12 < 210 > 4 < 211 > 12 < 212 > DNA < 213 > Home > sapiens 30 < 400 > 4 agtaaacaaa ca 12 < 212 > DNA < 213 > Homei saj) iens < 400 > 5 40 gcttcattcc gaatcaccag 20 < 210 > 6 < 211 > 20 < 212 > DNA 45 < 213 > Homo sapiens < 400 > 6 gtcccctccc tccaactatc 20

Claims (60)

1. CLAIMS 1. A method of diagnosing a mammal with regard to the increased possibility of developing an eye disease, said method comprising analyzing nucleic acids from said mammal to determine whether said nucleic acids contain a mutation in a FREAC3 gene, where the presence of said mutation is an indication that said mammal has an increased possibility of developing an eye disease.
2. 2. The method of claim 1, wherein said mammal is a human.
3. 3. The method of claim 1, wherein said disease is glaucoma.
4. 4. A method of diagnosing a mammal with regard to the increased possibility of having a developmental defect, said method comprising analyzing nucleic acids from said mammal to determine if said nucleic acids contain a mutation in a FREAC3 gene, where the presence of said mutation it is an indication that said mammal has an increased possibility of having a developmental defect.
5. The method of claim 1 or 4, wherein said mutation is a nonsense mutation.
6. The method of claim 5, wherein said nonsense mutation is a transversion from G to C in the coding nucleotide 245, wherein said transversion results in a Ser82Thr mutation in the helix 1 of the FREAC3 front domain.
7. The method of claim 5, wherein said nonsense mutation is a transversion from G to C in coding nucleotide 261, wherein said transversion results in an Ile87Met mutation in helix 1 of the FREAC3 front domain.
8. The method of claim 1 or 4, wherein said mutation is a frame shift mutation.
9. The method of claim 8, wherein said frame shift mutation results from a deletion of ten base pairs of coding nucleotides 93 through 102.
10. The method of claim 8, wherein said mutation results a truncated protein.
11. The method of claim 1 or 4, wherein primers are used to detect said mutation.
12. The method of claim 11, wherein said primers used to detect said mutation are selected from the primers shown in Table 1.
13. 13. The method of claim 1 or 4, wherein said method further comprises the step of sequencing nucleic acids. encoding FREAC3 from said mammal.
14. The method of claim 13, wherein said method further comprises the step of using nucleic acid primers for the FREAC3 gene, and wherein said primers are used for DNA amplification by the polymerase chain reaction.
15. The method of claim 1, wherein said analysis includes detecting the loss of a recognition site for a restriction endonuclease.
16. 16. The method of claim 15, wherein said restriction endonuclease is Alu I.
17. The method of claim 1 or 2, wherein said analysis includes detecting the gain of a recognition site for a restriction endonuclease.
18. 18. The method of claim 17, wherein said restriction endonuclease is Bsp Hl.
19. The method of claim 1 or 4, wherein said analysis includes detecting a loss of one or more nucleotides.
20. The method of claim 1 or 4, wherein said analysis includes detecting a gain of one or more nucleotides.
21. 21. The method of claim 1 or 4, wherein said analysis includes mismatch detection.
22. 22. The method of claim 21, wherein said analysis includes single filament conformational polymorphism (SSCP) analysis.
23. 23. The method of claim 21, wherein said analysis includes restriction fragment length polymorphism (RFLP) analysis.
24. 24. A kit for FREAC3 nucleic acid analysis, said kit comprising nucleic acid probes for analyzing the nucleic acid of a mammal, wherein said analysis is sufficient to determine if the mammal contains a mutation in said FREAC3 nucleic acid.
25. 25. A method of making an antibody that specifically binds a mutant FREAC3 polypeptide, said method comprising administering a mutant FREAC3 polypeptide, or fragment thereof, wherein said administration is to an animal capable of generating an immune response, and isolating said antibody from said animal.
26. 26. A method of detecting the presence of a mutant FREAC3 polypeptide, said method comprising reacting a sample with an antibody that specifically binds to a mutant FREAC3 polypeptide and testing the binding of said antibody to said mutant polypeptide.
27. The method of claim 26, wherein said mutant FREAC3 polypeptide has a threonine residue at position 82 of the amino acid FREAC3.
28. The method of claim 26, wherein said mutant FREAC3 polypeptide has a methionine residue at position 87 of the amino acid FREAC3.
29. 29. The method of claim 26, wherein said mutant FREAC3 polypeptide has an amino acid sequence that differs from the wild type sequence of FREAC3, where said amino acid sequence differs from carboxy terminal to amino acid 33 of FREAC3 (ala 33).
30. 30. A method of diagnosing a mammal in terms of • the increased possibility of developing an eye disease, said method comprising detecting the presence of a mutant FREAC3 polypeptide in said mammal, wherein the presence of said mutant FREAC3 polypeptide indicates that said mammal has a mutation in a FREAC3 gene, where the presence of said mutation is an indication that said mammal has an increased possibility of developing an eye disease.
31. 31. A method of diagnosing a mammal with regard to an increased possibility of having a developmental defect, said method comprising detecting the presence of a mutant FREAC3 polypeptide in said mammal, wherein the presence of said mutant FREAC3 polypeptide indicates that said mammal has a mutation in a FREAC3 gene, where the presence of said mutation is an indication that said mammal has an increased possibility of having a developmental defect.
32. 32. The method of claim 1, 4, 30 or 31, wherein said mammal is pre-natal.
33. 33. The method of claim 1, 4, or 27-30, wherein said mammal is a human.
34. 34. A kit for FREAC3 nucleic acid analysis, said kit comprising antibodies for analyzing polypeptides of a mammal, wherein said analysis is sufficient to determine whether said mammal contains a mutation in said FREAC3 nucleic acid.
35. 35. Nucleic acid encoding FREAC3 mutant, wherein said nucleic acid has at least one mutation, wherein said mutation is an indication that a mammal from which said nucleic acid is derived has an increased possibility of developing glaucoma.
36. 36. The mutation of claim 35, wherein said mutation is a transversion from G to C in the coding nucleotide 245.
37. 37. The mutation of claim 35, wherein said mutation is a transversion from G to C in the coding nucleotide. 261.
38. 38. The mutation of claim 35, wherein said mutation is a deletion of nucleotides 93 to 102.
39. 39. The nucleic acid of claim 35, wherein said nucleic acid is operably linked to regulatory sequences for expression of said polypeptide, and wherein said regulatory sequences comprise a promoter.
40. 40. A cell containing the nucleic acid of claim 39.
41. 41. The cell of claim 40, wherein said cell is a prokaryotic cell.
42. 42. The cell of claim 40, wherein said cell is a eukaryote cell.
43. 43. The cell of claim 42, wherein said cell is a yeast cell. •
44. 44. The cell of claim 42, wherein said cell is a mammalian cell.
45. 45. The promoter of claim 39, wherein said promoter is inducible.
46. 46. A transgenic non-human mammal, containing the nucleic acid of claim 39.
47. 47. The mammal of claim 46, wherein said mammal is a rodent.
48. 48. The transgenic mammal of claim 46, wherein one or both of the endogenous alleles encoding a FREAC3 polypeptide are disturbed, deleted, or otherwise nonfunctional.
49. 49. Transgenic mammalian cells of claim 46.
50. 50. A non-human mammal, wherein one or both of the endogenous alleles encoding a FREAC3 polypeptide are mutated in positions corresponding to those of Figure 2.
51. 51. Mammalian cells of claim 50
52. 52. A method of detecting a compound useful for the prevention or treatment of an eye disease, said method comprising assaying the transcription levels of a reporter gene operably linked to a promoter, said promoter comprising a FREAC3 ligation site. , said method comprising the steps of: (a) exposing said reporter gene to said compound, and (b) assaying said reporter gene in terms of an alteration in the activity of the reporter gene in relation to a reporter gene not exposed to said compound.
53. 53. The method of claim 52, wherein said reporter gene is in a cell.
54. 54. The method of claim 53, wherein said '• cell is in an animal.
55. 55. The method of claim 52, wherein an increase in said transcription indicates a compound useful for the prevention or treatment of glaucoma.
56. 56. The method of claim 1, 30 or 52, wherein said eye disease is glaucoma.
57. 57. A method of treating an eye disease by gene therapy in vivo, said method comprising introducing into the cells of the eye a nucleic acid encoding wild-type FREAC3, wherein said nucleic acid is operably linked to regulatory sequences for expression of said FREAC3, wherein said regulatory sequences comprise a promoter, and wherein said expression of said FREAC3 is sufficient to ameliorate the symptoms of said disease.
58. 58. The method of claim 57, wherein said nucleic acid is introduced into said cells by means of a viral vector, wherein said vector contains said nucleic acid encoding said FREAC3.
59. 59. The method of claim 57, wherein said nucleic acid is introduced into said cells by transformation.
60. 60. The method of claim 4, wherein said developmental defect is a cardiac defect. Res men The invention presents novel mutations in the FREAC3 gene. The finding provides methods for the early diagnosis of glaucoma, other eye disorders, and heart defects. Also provided are cells that have at least one FREAC3 gene deficient. Such cells can be used to detect therapeutic compounds that mimic FREAC3, are FREAC3 agonists, or otherwise modulate the level of biological activity of FREAC3.