JP2008142031A - Method for testing congenital eye disease comprising detecting mutation of wfdc1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for testing by which a causative substance (a gene or a protein) of congenital eye diseases in a mammal can be specified and the congenital eye diseases can be tested by utilizing the gene or the protein, and to further provide a reagent utilized in the method for testing and a kit for testing the congenital eye diseases. <P>SOLUTION: The method for testing, by which a mutation of the gene or the protein of WFDC1 (WAP four disulfide core domain 1) that is one cause of the congenital eye diseases is tested, is used. Specifically, the mutation contained in the WFDC1 contained in a nucleic acid sample is analyzed by directly confirming the sequence or directly subjecting the resultant amplified fragment to electrophoresis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a test method for a congenital eye disease, which is one of mammalian genetic diseases, and specifically to a test method for detecting a mutation in the gene or protein of WFDC1. Furthermore, the present invention relates to a primer or primer set used in these inspection methods, a test reagent containing them, and a test kit.

  In recent years, genetic diseases have been highlighted as a major problem not only in humans but also in domestic animals. Genetic diseases are a problem that cannot be ignored for the development of the livestock industry, for example. The most effective method for solving a genetic disease that adopts an autosomal single recessive genetic mode is to identify a disease-causing gene and its mutation and establish a genetic diagnosis method for detecting a carrier individual. In hereditary diseases that are dominated by autosomal recessive genes, apparently normal individuals appear by mating with normal carriers, so it is necessary to efficiently detect individual carriers and remove them from the population to prevent the onset of the disease. Is extremely important. Generally, when an autosomal recessive gene becomes homozygous and an affected individual appears, it is considered that a considerable number of carrier individuals are prevalent in the population. For example, in the case of domestic cattle and other livestock such as cattle that tend to be mated in such a way that a large number of individuals are derived from a single bull by artificial insemination and the inbreeding coefficient is relatively high The effect is serious (Non-Patent Document 1).

  Livestock, pets, racehorses, etc. often place great importance on pedigree in order to produce highly capable items. For example, currently registered livestock includes dairy cattle, beef cattle, and pigs. Further, even humans may suffer from congenital eye diseases, and in such cases, if genetic analysis is possible, measures can be taken regarding the onset of congenital eye diseases.

  The development of mammalian eyeballs is a very complex process, and it has been clarified that genes such as PAX6 and CHX10 play an important role in the development of eyeballs. In other words, if any of these genes is abnormal, it causes congenital eye disease. However, even if these genes are normal, they may suffer from congenital eye diseases. The cause of congenital eye disease is considered to have a genetic cause in addition to the genes shown above.

  As one means for preventing congenital eye diseases, a method of avoiding the use of a carrier of a gene causing the disease for mating can be considered. To do so, it is necessary to diagnose existing congenital eye diseases at the genetic level and to quickly find a carrier for the disease gene. If genes related to congenital eye diseases can be found in addition to the genes such as PAX6 and CHX10, congenital eye diseases can be prevented more reliably.

There is a report analyzing genes related to congenital eye diseases other than the above (Non-patent Document 2). Here, with regard to cattle, chromosomal mapping of the grandfather 1, sires 3, mother 9 and their offspring is performed for phenotypes of eye diseases and genes associated with the diseases, and congenital eyes It has been reported that the region of the chromosome related to the disease has been confirmed. However, we have not yet identified the specific genes and their mutation status.
Clinical Veterinary Vol.20, No.2 (2002) Mammalian Genome. 2005 Sep; 16 (9): 731-737

  An object of the present invention is to provide a test method capable of identifying a causative substance (gene or protein) of a congenital eye disease in a mammal and testing the congenital eye disease using the gene or protein. Furthermore, this invention makes it a subject to provide the reagent and congenital eye disease test | inspection kit which are utilized for this test | inspection method.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that WFDC1 gene and protein mutations are one of the causes of congenital eye diseases, and the WFDC1 gene or protein mutations By detecting this, it has been found that carriers can also be detected, and the present invention has been completed.

That is, this invention consists of the following.
1. A method for examining a congenital eye disease, comprising detecting a mutation of WFDC1 in a mammal.
2. 2. The test method according to item 1 above, wherein the mutation of WFDC1 detects a mutation of a gene encoding WFDC1 or a mutation of WFDC1 protein.
3. The inspection method according to 1 or 2 above, which comprises the following steps a) to c):
Step a) obtaining a mammalian nucleic acid sample;
Step b) A step of subjecting the nucleic acid sample obtained in step a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a mammalian WFDC1 gene is amplified;
Step c) A step of examining the presence of WFDC1 mutation using the amplified nucleic acid fragment obtained in step b).
4). In any one of the preceding items 1 to 3, the method for testing WFDC1 is a method selected from any of electrophoresis, nucleic acid chip method, immunological method, and restriction fragment length polymorphism (RFLP) detection method Inspection method described.
5. 5. An oligonucleotide having a primer function used for obtaining an amplified nucleic acid fragment in the test method according to item 4 above, comprising 15 to 35 nucleotides selected from the base sequence of a mammalian WFDC1 gene An oligonucleotide capable of amplifying a region containing a mutation site associated with congenital eye disease in the WFDC1 gene.
6). 6. The oligonucleotide according to item 5 above, wherein the mammalian WFDC1 gene is any WFDC1 gene selected from a mammal that can be a domestic animal, a pet, a competitive animal, a laboratory animal, and a human.
7). The oligonucleotide according to item 5 or 6, which is selected from the following 1) to 3):
1) An oligonucleotide comprising 15 to 35 nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and capable of amplifying a region containing a mutation site associated with congenital eye disease in the WFDC1 gene;
2) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 4 or 5 in the sequence listing;
3) A complementary strand of the oligonucleotide according to 1) or 2) above.
8). 8. A primer set consisting of at least two types of oligonucleotides selected from the oligonucleotides according to any one of 5 to 7, wherein the at least two types of oligonucleotides are associated with congenital eye diseases in the WFDC1 gene. A set of primers characterized in that a region containing a mutation site can be amplified.
9. A reagent for examination of congenital eye disease by detecting a mutation of WFDC1, comprising the oligonucleotide according to any one of 5 to 7 above.
10. A kit for testing a congenital eye disease by detecting a mutation in WFDC1, comprising the set of primers set forth in item 8 above.

  According to the test method of the present invention, it is possible not only to detect carriers but also to early diagnosis of an individual who has developed a congenital eye disease that can be caused by a mutation in the gene or protein of WFDC1. As a result, the occurrence of congenital eye diseases can be prevented more reliably.

  Congenital eye diseases associated with WFDC1 mutations are often recessive, and are expressed only when the WFDC1 gene has a homozygous mutation, and only one of the genes is a mutated carrier. Often does not represent congenital eye disease. However, when a carrier and a carrier are crossed, a born child may have a homotypic gene and may express a congenital eye disease. In addition to examining the carrier, it is possible to diagnose the affected individual at an early stage, and more reliably prevent the occurrence of congenital eye diseases.

  A method for examining a congenital eye disease by detecting a mutation of a WFDC1 gene or protein in a mammal according to the present invention will be described below. In the present invention, the congenital eye disease refers to a congenital eye disease like letters.

  In the present invention, WFDC1 refers to WAP four disulfide core domain 1 and is also called Prostates stromal protein ps20 (PS20). The WFDC1 gene has been identified and reported as a gene that is strongly expressed in prostate cancer-derived cells, and has so far attracted attention as a gene that may be mainly involved in the development of cancer mainly in prostate cancer. It was. However, the relationship between congenital eye disease and WFDC1 was first found in the present invention.

  In the present invention, mammals are not particularly limited, and examples include mammals that can be domestic animals, pets, competitive animals, and laboratory animals in addition to humans. For example, in the case of livestock and pets, a congenital eye disease based on a mutation in the WFDC1 gene can be prevented by detecting carriers by genetic testing and avoiding mating between carriers. In the case of experimental animals, a congenital eye disease model typified by animals such as mice and rats is established by using transgenic animals, knockout animals, drug-induced mutant animals, and the like in which the WFDC1 gene is mutated. be able to. Furthermore, it is also conceivable to perform genetic testing in humans in order to eliminate genetic diseases.

  The base sequence of the WFDC1 gene of the present invention is not particularly limited as long as it is the sequence of the WFDC1 gene of each mammal exemplified above, but can be appropriately selected depending on the target object of the test. Specifically, in addition to humans, target animals such as cattle, horses, goats, pigs, sheep, and other livestock, mice, rats, monkeys, and other experimental animals can be selected according to the purpose. Mutation can be detected based on the base sequence of the WFDC1 gene. More specifically, the base sequence of the bovine WFDC1 gene can be represented by, for example, GenBank Accession No. NW — 929284, and the mRNA sequence can be represented by GenBank Accession No. XM — 581642.

  In the present invention, the mutation of WFDC1 is not particularly limited. For example, a specific nucleotide in the base sequence of the WFDC1 gene may be mutated or an amino acid in the WFDC1 protein may be mutated. For example, it may be considered that the mutation of a nucleotide at a specific position of a gene has some influence on the function of the protein, or the protein itself is not expressed. Furthermore, although the protein is expressed, the amount thereof may be insufficient.

  Therefore, although the position of the mutation of WFDC1 in the present invention is not particularly limited, for example, it may affect the synthesis of WFDC1 protein and the protein cannot be expressed sufficiently. Specifically, with regard to the gene encoding WFDC1 protein, one or more nucleotide substitutions, deletions, additions at the upstream site may be considered, or one or more nucleotides at sites other than the upstream site may be considered. There may be cases where nucleotides are inserted or deleted. These mutations may cause the WFDC1 gene itself to fail. Specifically, it may be considered that the protein synthesis ability is reduced due to inhibition of transcription or translation into mRNA, and that the frame shift and the end of protein synthesis are affected. Moreover, it cannot be denied that a specific amino acid is mutated due to a specific nucleotide mutation, regardless of the frame shift or the end of protein synthesis. Furthermore, although the protein is expressed, the amount thereof may be insufficient.

  As specific mutations related to congenital eye diseases, in the region of the second exon of the WFDC1 gene (SEQ ID NO: 2) shown in SEQ ID NO: 1 in the sequence listing, there are six consecutive cytosines in the normal WFDC1 gene ( In the WFDC1 gene of an individual expressing a congenital eye disease in the 67th to 72nd positions of the sequence shown in SEQ ID NO: 2, there can be mentioned a mutation in which seven cytosines are continuous (see FIG. 2). Specifically, there may be a case where a single nucleotide (cytosine) is inserted to cause a frame shift during protein synthesis and a protein having a completely different structure from a normal protein is synthesized (see FIG. 3). ).

  In the present invention, the test method in the case of testing based on a gene is specifically performed by a method including the following steps a) to c). Additional steps may be added as appropriate during or before and after the following steps a) to c).

Step a) obtaining a mammalian nucleic acid sample;
Step b) A step of subjecting the nucleic acid sample obtained in step a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a mammalian WFDC1 gene is amplified;
Step c) A step of examining the presence of WFDC1 mutation using the amplified nucleic acid fragment obtained in step b).

Step a) In the present invention, the nucleic acid sample refers to a sample containing, for example, genomic DNA, cDNA and / or mRNA. Samples containing such nucleic acids can be collected from any part of a mammal, such as blood, plasma, serum, tissue, body fluid (saliva, sweat, nasal discharge, semen, lymph, etc.), etc. Can do. The nucleic acid sample can be obtained by a general method known per se, for example, the method described in Molecular Cloning Laboratory Manual, Second Edition (T. Maniatis et al., Cold Spring Harbor Laboratory, 1989). it can.

About Step b) In the test method of the present invention, the nucleic acid sample obtained in Step a) can be subjected to a gene amplification reaction, and the amplified nucleic acid fragment can be used. As a method for the gene amplification reaction, a method known per se can be used, and it is not particularly limited as long as a region necessary for gene analysis can be amplified. For example, a PCR method, a nucleic acid amplification method using RNA polymerase (JP-A-2- 5864, JP-A-7-203999), strand displacement amplification method (JP-B-7-114718, JP-A-7-88242), LAMP method, LCR (ligase chain reaction) method The law can be used. Of these, the PCR method is preferably used.

  In step b), a region containing a mutation site that may be present in the WFDC1 gene is amplified by the above method to obtain an amplified nucleic acid fragment. In order to obtain such a nucleic acid fragment, it is necessary to use a primer capable of amplifying a region sandwiching the mutation site of the gene. In the present invention, the position of the mutation is not particularly limited.

  The primer that can be used is not particularly limited as long as it can amplify the sequence of the mammalian WFDC1 gene, and can be appropriately selected depending on the target object of the test. Specifically, in addition to humans, target animals such as cattle, horses, goats, pigs, sheep, and other livestock, mice, rats, monkeys, and other experimental animals can be selected according to the purpose. Mutation can be detected based on the base sequence of the WFDC1 gene.

  More specifically, for example, when detecting a mutation in the region of the second exon (SEQ ID NO: 2) in the base sequence shown in the base sequence of the bovine WFDC1 gene (SEQ ID NO: 1), the SEQ ID NO: in the sequence listing The oligonucleotide which consists of 15-35 nucleotides selected from the base sequence of the area | region containing the 2nd exon shown in 3 and has a primer function can be used as a primer. The complementary strand can also be used as a primer. Specifically, an oligonucleotide containing the base sequence shown in SEQ ID NO: 4 or 5 in the sequence listing can be used as a primer. Furthermore, the complementary strand of these oligonucleotides can also be used as a primer.

  The primer combination method to be used can be appropriately selected depending on the gene amplification method. For example, when the PCR method is used, specifically, a forward primer and a reverse primer can be used as a set of primer sets. For example, it comprises 15 to 35 nucleotides selected from the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, more preferably selected from the nucleotide sequence of SEQ ID NO: 3, and is a mutation associated with congenital eye diseases in the WFDC1 gene. A set of primer sets can be used as oligonucleotides that can amplify the region including the site. More specifically, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 4 and 5 in the sequence listing are used as a set of primer sets. Can be used. Furthermore, an oligonucleotide composed of a complementary strand of each of the oligonucleotides shown above can be used as a set of primer sets.

About Step c) In Step c), the nucleic acid fragment obtained in Step b) is analyzed to check for the presence of mutations. The method for detecting the presence of the mutation is not particularly limited, and can be applied to a test method known per se. For example, as an inspection method for WFDC1, electrophoresis method, nucleic acid chip method, restriction fragment length polymorphism (RFLP) detection method and the like can be exemplified. Furthermore, it can be analyzed by expressing by genetic manipulation based on the obtained nucleic acid fragment and identifying and quantifying the expressed substance. Identification of the expressed substance can be performed by Western blotting and the like, and quantification can be performed by immunological techniques such as ELISA.

  For example, in the electrophoresis method, the obtained amplified fragment can be directly subjected to electrophoresis, and a mutation can be detected from the degree of electrophoresis. In the nucleic acid chip method, analysis can be performed by a method known per se. Specifically, it is based on a hybridization method using an appropriate DNA fragment containing a mutation site as a probe. For example, mutations can be analyzed by reacting a DNA probe immobilized on a substrate with the above nucleic acid fragment and reacting the hybridized nucleic acid with a substance labeled with fluorescence, luminescence, radioisotope, or the like. . As an immunological technique, a method of reacting a protein expressed based on an amplified nucleic acid or a protein sample of a test subject animal with an anti-WFDC1 antibody or an anti-mutant WFDC1 antibody, such as a Western blotting method, can be used. Furthermore, a method of detecting and examining restriction fragment length polymorphism (RFLP) is also used.

  As another method, if there is a mutation at a specific site, a specific base sequence is incorporated so that it reacts with the restriction enzyme, so that the size of the nucleic acid fragment obtained when the corresponding restriction enzyme is allowed to act can be electrophoresed. Can be analyzed and analyzed. For example, in the region of the second exon of the WFDC1 gene (SEQ ID NO: 2) shown in SEQ ID NO: 1 in the sequence listing, a specific recognition sequence for the restriction enzyme BglI is obtained in the vicinity of the mutation site when a mutation occurs. A polynucleotide consisting of a base sequence is incorporated. After the region containing the relevant portion is amplified by a nucleic acid amplification method such as PCR using the primer set described in SEQ ID NOs: 6 and 7 in the sequence listing, a nucleic acid fragment is obtained and the restriction enzyme BglI is allowed to act Analysis can be performed by electrophoresis of the fragments (see FIG. 5).

  As another analysis method used in this step, a method for examining a single chain conformation polymorphism (SSCP) can also be used. This method is a method for examining the difference in higher order structure depending on the base sequence formed by single-stranded DNA due to intramolecular interaction as a difference in migration speed by electrophoresis (Proc. Natl. Acad. Sci). USA, 86, 2766-2770 (1989)). The presence or absence of mutation can be detected by subjecting the nucleic acid fragment obtained in step b) to, for example, electrophoresis and comparing the movement speed with that of a nucleic acid fragment derived from a normal individual. As a detection method other than the RFLP method and the SSCP method, a known mutation detection method such as the DDGE method can be used.

  By the WFDC1 inspection method of the present invention, not only an individual expressing a congenital eye disease but also a carrier can be inspected and detected. Therefore, the congenital eye disease referred to in the present invention means that it is genetically abnormal, and it should be interpreted in a broad sense including the carrier regardless of the presence or absence of symptoms. In particular, when a symptom is manifested, it refers to a congenital eye disease or a phenotype.

  Independent of gene analysis, mutations in the WFDC1 protein can also be examined directly. In this case, for example, blood, plasma, serum, tissue, body fluid (saliva, sweat, nasal discharge, semen, lymph, etc.), etc. are collected and the presence of WFDC1 protein is confirmed by an immunological technique such as ELISA. It can be inspected.

Test reagent The test reagent of the present invention is specifically a primer capable of amplifying a desired region of a gene contained in the nucleic acid sample, a buffer solution necessary for gene amplification, a reaction reagent, a restriction enzyme, and a detection reagent. (For example, a reagent for electrophoresis, a probe, etc.) In particular, specific primers for nucleic acid amplification in the present invention can be mentioned as the reagent of the present invention.

Congenital eye disease test kit The congenital eye disease test kit of the present invention specifically includes a set of primer sets that can amplify a desired region of a gene contained in the nucleic acid sample, and further includes a nucleic acid. Any one or more selected from buffers, reaction reagents, restriction enzymes, detection reagents (for example, electrophoresis reagents, probes, etc.) necessary for amplification can be included. In addition, there is no problem even if a device for inspection is included in the kit.

  Below, the background that led to the completion of the present invention will also be described, and then specific examples will be described with reference to examples.

  In order to analyze genes related to congenital eye diseases, according to the method of Non-Patent Document 1, nucleic acid samples were obtained from domestic cattle including about 20 affected individuals, and chromosome linkage analysis was performed. Nucleic acid samples were obtained from peripheral blood and serum by phenol / chloroform extraction.

  When linkage analysis was performed using 240 microsatellite markers covering all bovine chromosomes, strong linkage was observed between BMS1322 and BMS14 on chromosome 18. Therefore, it was revealed that the causative gene of congenital eye disease exists in the proximal part of chromosome 18. Furthermore, many new microsatellite markers around this area were developed, and the candidate region was narrowed by the homozygote mapping method, and finally it was possible to identify the region of about 1.1 Mb (Non-Patent Document 2, FIG. 1). reference).

  Each of the genes present in the region specified above was analyzed, and the present inventors found for the first time a relationship between a congenital eye disease and the WFCD1 gene. The contents will be described in detail below by examples.

  The present invention will be described below with reference to examples, but it is needless to say that the present examples are for the purpose of better understanding the contents of the invention, and the present invention is not limited to these examples.

Example 1 Confirmation of Mutant WFDC1 Gene 1) Amplification by PCR A portion of the region (SEQ ID NO: 3) containing the second exon was amplified from a bovine nucleic acid sample obtained by phenol / chloroform extraction from peripheral blood and serum. As can be seen, nucleic acids were amplified by PCR using the following forward and reverse primers.
Forward primer 5'-GTAGGCGGAGGAGGTAGGC (SEQ ID NO: 4)
Reverse primer 5'-GTAGGCGCAGCCGTTGTAG (SEQ ID NO: 5)

The following reagents were used in the PCR method. Genomic DNA of normal cows, congenital eye disease cows, and their mother cows were used as templates.
Template DNA (10ng) 1μ1
2 × GC buffer I (TAKARA) 5 μl
dNTPs 1μ1
Primer (20 μM each) 0.25 μl each
Taq 0.05U
H ₂ O total amount 100 μl

  PCR was performed at 94 ° C. for 5 minutes, followed by 30 cycles of 94 ° C. for 30 seconds, 59 ° C. for 30 seconds, and 72 ° C. for 45 seconds, then held at 72 ° C. for 7 minutes and stored at 4 ° C. .

2) Analysis of base sequence by cloning The amplified DNA fragment obtained from 1) above was recovered from the PCR reaction solution, the amplified DNA fragment was cloned into a plasmid vector, and the base sequence was determined by the dideoxy method.

  As a result, insertion of one base was confirmed in the second exon region (SEQ ID NO: 2) of the WFDC1 gene. That is, in the normal individual, the sequence in which the C in the 67th to 72nd positions of SEQ ID NO: 2 was repeated 6 times had 7 C repeats in the affected individual (see FIG. 2). As a result of this insertion of one base, a frame shift is caused when a WFDC1 protein is synthesized from a mutant WFDC1 gene, and the amino acid sequence after the 67th amino acid residue is normal (wild type (SEQ ID NO: 10)). It was completely different and it was confirmed that the 126th early stop codon appeared (see FIG. 3 (SEQ ID NO: 11)). Therefore, it was considered that the WFDC1 protein produced from the mutant WFDC1 gene completely lost the function of the normal WFDC1 protein.

(Example 2) Inspection method by direct electrophoresis The amplified DNA fragment obtained by the PCR method of Example 1 was recovered from the PCR reaction solution, and the DNA fragment was directly subjected to electrophoresis. The results are shown in FIG. Bands were observed at different positions in normal cattle and in cattle with a carrier or eye disease phenotype (Affected).

  From the above results, it was confirmed that a normal bovine, carrier or eye disease phenotype (Affected) gene could be examined by direct electrophoresis.

(Experimental example 3) Inspection method by insertion of restriction enzyme recognition sequence In the second exon region of the WFDC1 gene, using a mismatch primer, the restriction enzyme BglI recognition sequence (5'-GCCNNNNNGGC) is used in the vicinity of the mutation site. : A polynucleotide consisting of a specific base sequence (GCCCCC: SEQ ID NO: 9) was incorporated so as to obtain SEQ ID NO: 8). A region including the corresponding portion was amplified by a nucleic acid amplification method by PCR using the primer sets described in SEQ ID NOs: 7 and 8 in the sequence listing to obtain nucleic acid fragments. The obtained amplification product was analyzed by electrophoresis after the restriction enzyme BglI was allowed to act (see FIG. 5). Theoretically, when a mutation was observed, it was cleaved with a restriction enzyme, and when normal, it was not cleaved with a restriction enzyme (see FIG. 6).

Forward primer 5'-GTAGGCGGAGGAGGTAGGC (SEQ ID NO: 6)
Reverse primer 5'-GCGTGCAGCCTG GCAGGCGCCGG (SEQ ID NO: 7)

  When the nucleic acid sample of the example was confirmed, as shown in FIG. 7, a band was observed at 98 bp in the case of normal cattle, and a band was observed at 76 bp in the ophthalmic disease phenotype, and almost theoretical results could be obtained. . In addition, two bands were observed in the carrier, and the carrier having the mutant WFDC1 gene could be confirmed.

  From the above results, it was confirmed that a normal bovine, carrier, or eye disease phenotype (Affected) gene can be examined by a restriction enzyme recognition sequence insertion method using a mismatch primer.

  As described above, according to the test method of the present invention, it is possible not only to detect a carrier but also to early diagnosis of an individual who has developed a congenital eye disease that can be caused by a mutation in a WFDC1 gene or protein. Become. As a result, the occurrence of congenital eye diseases can be prevented more reliably.

  Congenital eye diseases associated with WFDC1 mutations are often recessive, and are expressed only when the WFDC1 gene has a homozygous mutation, and only one of the genes is a mutated carrier. Often does not represent congenital eye disease. However, when a carrier and a carrier are crossed, a born child may have a homotypic gene and may express a congenital eye disease. In addition to examining the carrier, it is possible to diagnose the affected individual at an early stage, and more reliably prevent the occurrence of congenital eye diseases.

  Furthermore, a congenital eye disease model represented by an animal such as a mouse or a rat can be established by using a transgenic animal, a knockout animal, a drug-induced mutant animal, or the like in which the WFDC1 gene is mutated.

It is a figure which shows a chromosome map. It is a figure which shows an analysis result. (Example 1) It is a figure which shows the influence which acts on the synthesis | combination of WFDC1 protein by the variation | mutation of WFDC1 gene. It is a figure which shows the result by a direct electrophoresis method. (Example 2) It is a figure which shows the inspection method of the variation | mutation using a restriction enzyme (Example 3). It is a figure which shows the length of the obtained polynucleotide about what cleaved the amplification product with the restriction enzyme (BglI). (Example 3) It is a figure which shows the result by Example 3. FIG.

Claims (10)

A method for examining a congenital eye disease, comprising detecting a mutation of WFDC1 in a mammal. The test method according to claim 1, wherein the mutation of WFDC1 is performed by detecting a mutation of a gene encoding WFDC1 or a mutation of WFDC1 protein. The inspection method according to claim 1 or 2, comprising the following steps a) to c):
Step a) obtaining a mammalian nucleic acid sample;
Step b) A step of subjecting the nucleic acid sample obtained in step a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region containing a mutation site that may be present in a mammalian WFDC1 gene is amplified;
Step c) A step of examining the presence of WFDC1 mutation using the amplified nucleic acid fragment obtained in step b). The method for testing WFDC1 according to any one of claims 1 to 3, wherein a method selected from any of electrophoresis, nucleic acid chip method, immunological technique, and restriction fragment length polymorphism (RFLP) detection method is used. Inspection method described in 1. An oligonucleotide having a primer function used for obtaining an amplified nucleic acid fragment in the test method according to claim 4, wherein the oligonucleotide has 15 to 35 nucleotides selected from the nucleotide sequence of a mammalian WFDC1 gene The oligonucleotide which can amplify the area | region containing the site | part of the variation | mutation relevant to the congenital eye disease in WFDC1 gene. 6. The oligonucleotide according to claim 5, wherein the mammalian WFDC1 gene is any WFDC1 gene selected from mammals that can be domestic animals, pets, competitive animals, laboratory animals, and humans. The oligonucleotide according to claim 5 or 6, which is selected from the following 1) to 3):
1) An oligonucleotide comprising 15 to 35 nucleotides selected from the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and capable of amplifying a region containing a mutation site associated with congenital eye disease in the WFDC1 gene;
2) an oligonucleotide comprising the base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing;
3) A complementary strand of the oligonucleotide according to 1) or 2) above. A primer set consisting of at least two kinds of oligonucleotides selected from the oligonucleotide according to any one of claims 5 to 7, wherein the at least two kinds of oligonucleotides are associated with congenital eye diseases in the WFDC1 gene. A set of primers characterized in that the region containing the mutation site to be amplified can be amplified. The reagent for a test | inspection of the congenital eye disease by detecting the variation | mutation of WFDC1 containing the oligonucleotide of any one of Claims 5-7. A kit for testing a congenital eye disease by detecting a mutation in WFDC1, comprising the primer set according to claim 8.