WO2001088120A1

WO2001088120A1 - Gene associating open-angle glaucoma including normal ocular tension glaucoma

Info

Publication number: WO2001088120A1
Application number: PCT/JP2001/004067
Authority: WO
Inventors: Yukio Hattori; Ryo Suzuki
Original assignee: Tsubota Ltd.
Priority date: 2000-05-17
Filing date: 2001-05-16
Publication date: 2001-11-22
Also published as: AU2001258753A1; JP2002306165A

Abstract

Polynucleotides having substitution of thymine (T) by cytosine (C) at the -153 position of myocilin gene are identified as glaucoma-associated gene. To detect these polynucleotides, use can be made of primers of SEQ ID NOS:3 to 6 and probes of SEQ ID NOS:7 and 8. Also, kits with the use of the primers of SEQ ID NOS:3 to 6 or the probes of SEQ ID NOS:7 can be provided. Use of these kits makes it possible to detect normal ocular tension glaucoma and open-angle glaucoma at a much higher frequency than in a case with the use of glaucoma-associated genes reported hitherto. Also, these kits are usable in screening glaucoma in patients with a fear of hereditary glaucoma or asymptomatic carriers. Moreover, it becomes possible for the first time to diagnose open-angle glaucoma including normal ocular tension glaucoma prior to the onset.

Description

Genes related to open-angle glaucoma including normal tension glaucoma

The present invention relates to genes associated with open-angle glaucoma, including normal tension glaucoma. The present invention further relates to a primer or probe for detecting a glaucoma-related gene, a kit for detecting a glaucoma-related gene, a glaucoma pre-onset diagnostic kit, and a method for preliminary detection of glaucoma. Glaucoma is one of the major causes of blindness in Japan, Europe and the United States. In Japan, glaucoma including normal-tension glaucoma has been found in more than 3% of those aged 40 or older. Currently, about 200,000 people in Japan and about 800,000 worldwide have glaucoma. In the statistics of developed countries as a whole, it is the second leading cause of blindness, and its proportion is increasing with aging (Quigley, HA ヽ N. Engl. J. Med. 1998; 338: 1063- See 1064). Glaucoma generally occurs when the intraocular pressure is higher than the normal intraocular pressure, thereby impairing the blood circulation of the optic nerve tissue and causing optic nerve fibers to be exhausted, shed, and degenerated. Glaucoma is a serious eye disease that causes visual field abnormalities that can eventually lead to blindness. In Japan, normal tension glaucoma is characteristic, and there are more than three times as many patients as so-called high tension glaucoma.

Early detection is important for glaucoma treatment. Currently, glaucoma testing requires multiple examinations of intraocular pressure, visual field, and fundus such as optic disc and optic nerve fibers. However, since intraocular pressure in glaucoma fluctuates even in a short time, diagnosing normal tension glaucoma requires time and effort, such as performing intraocular pressure measurement over a day. In addition, patients often do not notice glaucoma even if their symptoms are advanced because the healthy eye supplements the visual field of the affected eye.

There are various types of glaucoma, but in recent years, genes associated with primary open-angle glaucoma (P0AG) have been identified. This gene is located on the long arm of chromosome 1 and is reported as the juvenile open-angle glaucoma gene (GLCL1A). It encodes a 54 kd protein called myocilin (MYOC) (see Stone, EM et al., Science 1997; 275: 668-670).

A number of mutations in the myocillin gene associated with glaucoma have been reported by several research groups (eg, Stone, E. Μ · et al., Science 1997; 275: 668-670; Rozsa, FW et al., Mol. Vis. 1998; 4:20, Kubota, R. et al., Biochem. Biophys. Res. Co Thigh. 1998; 242: 396-400, Williams-Lyn, D. et al. Can. J. Ophthalmol. 2000; 35: See 12-17). Most of these mutations occur in exons. Twenty-two amino acid mutations have been confirmed in young angle glaucoma, including those presented at academic conferences. However, the sum of all those known can only explain about 3% of open-angle glaucoma (Alward, WL et al., N. Engl. J. Med. 1998; 338: 1022- 1027; Z. Zhou et al., Hum. Mol. Genet. 1999; 8: 2221-2228). In myosin exons, five different alleles have been identified in Japanese patients (see Suzuki et al., Am. J. Hum. Genet. 1997; 61: 1202-1204), but the frequency of these alleles is high. Is also very small.

As a mutation in the promoter region, a substitution of guanine (G) to adenine (A) at position -83 has been reported (Fingert, JH et al., Hum. Mol. Genet. 1999; 8: 899). -See 905). However, this mutation is only a polymorphism and the disease specificity is very low. Nor was there a gene search for normotensive glaucoma, which has many patients. Furthermore, mutations in the promoted region of the myosillin gene have not been studied at all, although there have been reports of polymorphisms. Thus, the reported myocillin abnormalities can only detect a small portion of glaucoma (Williams-Lyn, D. et al., Can. J. Ophthalmol. 2000; 35: 12-17, Pang, C.P. Et al., Hum. Mutat. 2000; 15: 122).

In glaucoma, clinical diagnosis and ophthalmic treatment of intraocular pressure, visual field and fundus have developed. However, current medicine makes it difficult to restore visual function in advanced glaucoma. Therefore, early detection, prevention and treatment of glaucoma can be effective if people who have no current symptoms but have glaucoma or who are likely to have glaucoma in the future can be distinguished from those who are less likely to have glaucoma in the future. Should be done.

In addition, normal tension glaucoma, which is common in Japan, has normal intraocular pressure and no subjective symptoms. It is often overlooked even if you visit a department. No gene has been reported for this normal tension glaucoma.

Departure TO

The present inventors compared the nucleotide sequence of the promoter region of the myosin gene of a patient with open-angle glaucoma including normal-tension glaucoma with the nucleotide sequence of the corresponding portion of a healthy subject, and identified the promoter region of the patient with open-angle glaucoma. It was found that mutation occurred at a significant frequency at the position.

The gene according to the present invention is present in the promoter region of the myocillin gene, and has a remarkably high detection frequency in open-angle glaucoma including normal tension glaucoma. In this respect, the gene of the present invention is clearly different from the known mutations described above. Glaucoma often develops beyond the age of 40, but it is extremely difficult to make a definitive diagnosis early.

Therefore, by examining mutations in the promoter region revealed for the first time in the present invention, it plays an auxiliary diagnostic role to find a person who is likely to develop open-angle glaucoma including normotensive glaucoma. be able to. And it may be possible to make an effort to detect glaucoma early by conducting regular medical examinations for people with this sudden mutation.

By examining the presence or absence of this mutated gene associated with open-angle glaucoma, including normal tension glaucoma, it is possible to infer whether progeny that are susceptible to glaucoma are inherited in offspring. Further, as described below, the glaucoma-related gene according to the present invention has a much higher frequency of occurrence in open-angle glaucoma including normal-tension glaucoma than the previously reported glaucoma-related gene. We have found that

That is, the present invention relates to a polynucleotide comprising a DNA sequence represented by SEQ ID NO: 1, which is frequently present in open-angle glaucoma patients including normal-tension glaucoma in the promoter region of the myosin gene.

In this specification, the term “polynucleotide” is used in a broad sense including what is generally called “oligonucleotide”.

Further, the present invention relates to a polynucleotide in which thymine (T) at position −153 in the DNA sequence upstream of the human myosilin gene represented by SEQ ID NO: 2 is substituted with cytosine (C). The present invention relates to a polynucleotide comprising a part or all of a nucleotide, wherein the polynucleotide comprises a base at least at position -153. SEQ ID NO: 2 shows the nucleotide sequence at positions -473 to 3 of the human myocillin gene.

Here, the positions of the bases are as follows: in the DNA, the translation start point is the first position, the adjacent base upstream thereof is the −1 position, and n bases including the −1 position base are upstream by n bases. The position of the base located on the end side is called the 1 n-th position. For example, position -153 is located 153 bases upstream from the start of peptide synthesis; it is the position of the base in DNA. In the above-mentioned polynucleotide in which the -153 position of the myosillin gene is substituted with thymine (T) to cytosine (C), one or several bases are deleted, added or substituted at positions other than the -153 position. It may be.

In addition, the present invention can include fragments of any length, if necessary, as long as they include the -153 position as well as those containing the entire upstream region of the myosillin gene. For example, a polynucleotide having a base length of 19 to 3 16 bp can be exemplified. The present invention also relates to a primer used for amplifying the above-mentioned polynucleotide. As the primer, those having any base length and sequence can be used as long as the target polynucleotide is amplified. Preferably, a primer of SEQ ID NO: 3 or SEQ ID NO: 6 can be used. Also, a set of primers consisting of the primer of SEQ ID NO: 3 and any of the primers of SEQ ID NOs: 4 to 6 can be used.

Furthermore, the present invention relates to a kit for detecting the substitution of thymine (T) to cytosine (C) at position −153 of the promoter overnight region of the human myosillin gene, comprising the above primer. It can also provide kits containing such primers for diagnosing people who are more likely to develop glaucoma before developing glaucoma.These kits can be used to diagnose glaucoma, including normal tension glaucoma. If you are worried about the disease, perform an auxiliary diagnosis for those who are worried about the inheritance of the disease.

Further, the present invention provides a probe represented by SEQ ID NO: 7, which is capable of detecting that the position at position -153 of the promoter region of the myosillin gene is cytosine (C), and a position at position -153 of the promoter region of the human myosillin gene. Is a probe represented by SEQ ID NO: 8, which is capable of detecting thymine (T). These probes have a 5 ' Those labeled with tin, fluorescent or ³² P are preferred. Using these probes, it is possible to distinguish between the thymine (T) and the cytosine (C) at position -153 of the promoter region of the human myosillin gene.

The present invention also provides a method for detecting the substitution of thymine (T) to cytosine (C) at position −153 of the promoter region of the human myosillin gene, including the probes represented by SEQ ID NO: 7 and SEQ ID NO: 8. Regarding the kit. In addition, a kit for diagnosing a person who is likely to have glaucoma before developing glaucoma, comprising such a probe, can be provided. These kits provide an auxiliary diagnosis for those who are more likely to have open-angle glaucoma, including normotensive glaucoma.

Furthermore, the present invention provides a method for detecting glaucoma, which comprises detecting substitution of thymine (T) at position -153 with cytosine (C) in the DNA sequence of the promoter overnight region of the human myosillin gene. It relates to a method of detecting a highly likely person.

By using the primer, the probe, or the kit of the present invention, a mutation at the −153 base in the promoter region of the myosillin gene can be detected. As explained based on the following survey results, the substitution of thymine (T) to cytosine (C) at the -153 base in the promoter region of the myosillin gene is not restricted to glaucoma, especially in normal tension glaucoma. It is closely related to angle glaucoma. When glaucoma is discovered, a presymptomatic diagnosis (ie, future glaucoma) can be performed by examining whether the offspring or people in the family have a similar mutation. become. By detecting such a mutation, it is possible to detect glaucoma patients at an early stage and to detect a person who is likely to suffer from glaucoma with high accuracy. The offspring of the next generation are young, have not yet reached the age of onset of glaucoma, and it is not possible with current clinical techniques alone to determine whether they are healthy carriers or those who do not develop lifelong disease It is.

Violent form bear to launch

The present invention relates to polynucleotides having a glaucoma-associated mutation. Glaucoma that can be detected by the present invention includes normal-tension glaucoma and open-angle glaucoma. The glaucoma-related gene of the present invention is, specifically, a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 1, and a human represented by SEQ ID NO: 2 A polynucleotide comprising a part or the whole of a polynucleotide in which thymine (T) at position -153 in the DNA sequence upstream of the myosillin gene has been replaced by cytosine (C), wherein the polynucleotide comprises at least a base at position -153. It is about. The polynucleotide of the present invention includes a single-stranded DNA, a double-stranded DNA composed of a complementary strand thereof, and an antisense strand, and also includes a polynucleotide partially having a modified base. In addition, it includes those in which one or several bases are deleted, added or substituted at positions other than -153.

Such a polynucleotide is one of the genes responsible for glaucoma, especially normal tension glaucoma and open angle glaucoma. Therefore, primers, probes and kits for testing for the presence or absence of such a polynucleotide should be used in glandular diagnosis and glaucoma patients, and in genetic diagnosis for glaucoma progeny. Can be used.

In addition, the primer of the present invention is not particularly limited as long as it is a primer pair substantially complementary to a part of the upstream and downstream sequences at position −153 of the myosillin gene. Here, “substantially complementary” means that it is only necessary to be complementary enough to be able to anneal to the upstream and downstream sequences at position −153 of the myosillin gene. For example, a primer containing a sequence that is not complementary to a part of the myosillin gene at the 5 'end, or at the 3' end, or at both the 5 'end and the 3' end of the primer, as long as it can anneal to the myosillin gene. It is to be included in the primer of the present invention. Further, in order to prevent non-specific amplification or to introduce an appropriate restriction enzyme recognition site, a primer having a mismatch sequence that is not complementary to the myosillin gene can be used.

Examples of suitable primers include one or more primers of SEQ ID NO: 3 to SEQ ID NO: 6. Using a pair of primers consisting of the primer of SEQ ID NO: 3 and any one of SEQ ID NOs: 4 to 6, a polynucleotide containing the -153 position of the myosillin gene can be amplified by PCR or the like. .

The method for detecting the substitution of thymine (T) at position -153 of the myosin gene with cytosine (C) is not particularly limited. For example, the detection is performed by determining the base sequence by the Sanger method or the like. Or RFLP (Restriction Fragment Length As in the case of Polymorphism), it may be detected indirectly without determining the sequence. Restriction enzymes that can be used for RFLP include SinaI.

Furthermore, using the primer of the present invention for amplifying a polynucleotide, PCR (Polymerase Chain Reaction) ^ and PCR-RFLP (PCR-Restriction Fragment Length Length), PCR-SS0 (PGR-Specific Sequence) Oligonucleotide), PCR—SSCP (PCR-Single Strand Conformation Polymorphism analysis), allele-specific PCR (allele-specific PCR), etc., from thymine (T) to cytosine (C) at position -153 of the myosillin gene Replacement can be detected.

Further, the substitution of thymine (T) to cytosine (C) at position −153 of the myosin gene is detected by dot blot hybridization using the probe of the present invention. be able to. The probe of SEQ ID NO: 7 allows detection of the myosin gene whose position at position -153 is cytosine (C), and the probe of SEQ ID NO: 8 allows detection of the myosillin gene whose position of -153 is thymine (T).

Among the above methods for detecting the substitution of thymine (T) to cytosine (C) at position -153 of the myosillin gene, PCR, PCR-SSCP, PCR-RFLP, doubly hybridisation, allele specific The outline of the target PCR is described below.

PCR;

A primer is added to the reaction buffer containing dNTP, and a thermostable DNA polymerase is further added. The cycle of denaturation, annealing, and extension is repeated to amplify the myosin gene containing position -153. The reaction buffer is, for example, 10 mM Tris-Cl (pH 8.9), 1.5 mM MgCl2, 80 mM KC1, 0.5 mg / ml BSA, 0.1% sodium cholate (0.1%). A buffer containing Triton X-100 can be used. However, the present invention is not limited to this reaction buffer, and any reaction buffer can be used. The denaturation, annealing, and extension cycle is typically 30 seconds to 1 minute 30 seconds at 94 ° C for denaturation, 30 seconds to 1 minute 30 seconds at 55-68 ° C for annealing, and 72 ° C for extension. 25 to 40 cycles are performed in 30 seconds to 5 minutes, but the invention is not limited to this range. For example, denaturation can be performed at 94 ° C for 1 minute, annealing at 62 ° C for 1 minute, and extension at 72 ° C for 2 minutes for 25 to 40 cycles. As the thermostable DNA polymerase to be used, a commercially available thermostable polymerase such as Taq polymerase or Tth polymerase can be used.

The primer can use a primer pair that is substantially complementary to a portion of the sequence upstream and downstream of position -153 of the myosillin gene, as previously described. Those skilled in the art will appropriately select the reaction buffer, reaction conditions, and primers according to the length of the primers used, the complementarity of the primers, or the various PCR modifications described below. Only the sequence can be amplified.

PGR-vSSCP:

-Amplify by PCR using a pair of primers flanking position 153. The obtained fragment is denatured, and the difference in the sequence at position −153 can be detected by the difference in the electrophoretic mobility in polyacrylamide electrophoresis. As primers used for PCR-SSCP, primers of SEQ ID NO: 3 and SEQ ID NO: 4 can be used.

PCR-R.FLP method:

When PCR is performed using the primers of SEQ ID NO: 3 and SEQ ID NO: 5, the resulting 185 bp PCR fragment contains the myosillin gene in which the position -153 is substituted with thymine (T) for cytosine (C). The positions -154 to -149 are CCCGGG, and the positions of -153 to -149 are CTCGGG for the myosin gene without substitution at position -153. Since only the sequence of CCCGGG is recognized and cleaved by the restriction enzyme Smal, treatment of the 185 bp fragment after PCR containing CCCGGG with Smal will generate a 163 bp fragment and a 22 bp fragment. Therefore, the 185 bp fragment after PCR was treated with Smal, and the presence or absence of the 163 bp fragment was detected by polyacrylamide gel electrophoresis or agarose gel electrophoresis. It can be determined whether or not it has been replaced with cytosine (C).

Allele-specific PCR (Al) el e-sped l c PCR):

Of a pair of primers one that sandwich the position -153, one primer - 153 of the designed such portions that correspond comes 3 ³ terminus, thymine (T) at position -153 mustard The upstream sequence of the myocillin gene having a substitution for tosin (C) can be detected. An example of such a set of primers is the primers of SEQ ID NO: 3 and SEQ ID NO: 6.

The primer of SEQ ID NO: 6 is used at the 3 'end of the primer to prevent nonspecific growth. There is a mismatch with cytosine (C) at the fourth base from adenine (A). Using such a set of primers, nested PCR, that is, a sequence, under strict PCR conditions, so that only those in which position -153 is substituted with cytosine (C) are amplified. The fragment amplified by PCR using the primers of No. 3 and SEQ ID No. 4 is preferably subjected to PCR using the primers of No. 3 and No. 6 as type III. When the primers of SEQ ID NO: 3 and SEQ ID NO: 6 were used, finally, only the upstream sequence of the myosillin gene having the substitution of thymine (T) to cytosine (C) at position -153 was amplified, A 185 bp PCR amplified fragment is obtained. This fragment can be detected by agarose gel electrophoresis or polyacrylamide electrophoresis.

De, sotobro, sotohybridization:

For example, the PCR fragment containing the -153 position amplified using SEQ ID NO: 3 and SEQ ID NO: 4 is spotted on a nylon membrane or the like after denaturation. After incubating the membrane in a prehybridization buffer (eg, containing 5x Denhardt's solution (Denhardt, s), 5x SS 0.1% SDS, 0.1mg / ml denatured dicin sperm DNA) , 5, end Piochin, ³² P, and a probe with SEQ ID NO: 7 labeled SEQ ID NO: 8 was added at etc. fluorescence, further Inkyube over preparative. After washing the membrane, autoradiography, color development by alkaline phosphatase, chemiluminescence, or fluorescence is used to detect whether hybridization has occurred. Those skilled in the art can change and select the hybridization temperature and washing conditions in order to prevent non-specific hybridization. For example, the hybridization temperature may be about 40 hours to about 50 degrees Celsius for about 2 hours, and tetramethylammonium chloride may be used for washing.

When the probe of SEQ ID NO: 7 and the hybridization were detected, it was judged that the myosin gene had a substitution of thymine (T) with cytosine (C) at position -153, and the probe of SEQ ID NO: 8 was detected. If only hybridization is detected, it is determined that the myosin gene has no substitution of thymine (T) to cytosine (C) at position -153.

The kit of the present invention includes a primer capable of amplifying the promoter region of the myosinillin gene including position -153 of the myosillin gene or a probe capable of specifically hybridizing by distinguishing the difference in the base at position -153. As a reagent included in the kit Can be appropriately changed by a method for detecting base substitution. For example, when the nucleotide sequence is determined by the Sanger method, ddATP, ddTTP, ddCTP, ddGTP and dATP, dTTP, dCTP, dGTP, DNA synthase, etc. are included in addition to the primers. When base substitution is detected by RFLP, an appropriate restriction enzyme, for example, one containing Smal can be mentioned.

Further, the kit of the present invention comprises a buffer and a washing solution suitable for detecting the substitution of thymine (T) for cytosine (C) at position -153 of the promoter region of the human myosillin gene. Is also good.

The glaucoma pre-onset diagnostic kit of the present invention is for detecting the substitution of thymine (T) for cytosine (C) at position −153 of the promoter region of the human myosillin gene. As an active ingredient. The glaucoma pre-onset diagnosis kit of the present invention is useful for early detection and treatment of glaucoma.

The DNA whose presence or absence of substitution is detected by the present invention can be obtained from human hair, blood, body fluid, saliva, cultured cells, excised tissue, and the like, and is not particularly limited. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Room example

The following first and second surveys were conducted on Japanese patients with open-angle glaucoma, including normal-tension glaucoma. The subjects of the examination were 83 patients with open-angle glaucoma, including normal-tension glaucoma, who had a total family history of glaucoma, and healthy subjects who were considered to have no eye disease because they had not visited an ophthalmology department 101 people. Both groups have similar structures such as age.

Specifically, the following method was used. Nal method (Wang Lli et al: Purification of genomic DNA from human whole blood by isopropanol -fractionation with concentrated Nal and SDS. Nucleic Acid Res., 22: 1774-) from 83 peripheral blood leukocytes of patients with glaucoma (P0AG) 1775, 1994). As a control, 101 abnormal healthy subjects were analyzed for the same gene abnormality. Information on the normal nucleotide sequence of the myocillin gene was obtained from GenBank accession numbers AB006686, AB006686S2, AB006686S3.

(DPC; In a 0.5 ml microtube, add H20 15.5〃1, 1.251 ^ (1? 4; «1, lOx reaction buffer (100 mM Tris-Cl (pH 8.9), 15 mM MgCl2, 800 mM KC1, 5 mg / ml BSA, 1% sodium cholate, 1 TritonX-100) at 2.5〃1 and two 50 pmol / 〃1 primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 at 0.5〃1, Add DNA (about 0.5 g), mix well, add 1 U / 1 Tth DNA polymerase (Toyobo) l〃l, and overlay 1 drop of mineral oil. Thus, the promoter overnight region of the myosin gene was amplified under the following conditions.

As a general rule, heat denaturation at 94 ° C for 1 minute before repetition is followed by 1 minute at 94 ° C for denaturation, 1 minute at 62 ° C for annealing, and 2 minutes at 72 ° C for extension (final round). (4 minutes) 30 times. However, for the fragment for RFLP, the fragment amplified by performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 was diluted 1000-fold to form type II, and the fragment of SEQ ID NO: 5, which is a mismatch primer, was used. Amplification was performed by nested PCR between the primer represented by SEQ ID NO: 3 and the primer represented by SEQ ID NO: 3.

(2) Single sale 櫳诰 winter type '! ¹ cow (SSCP: single strand nonforma.tion polymorphism): PCR fragment 3 obtained using the primers represented by SEQ ID NO: 3 and SEQ ID NO: 4 contains 90% formamide ( One milliliter of 20 mM EDTA, 0.1% BPB, and 0.1% xylene cyanol) were mixed, heat denatured at 90 ° C. for 5 minutes, and quenched on ice. Run this at 37 ° C, 300V for 4 hours at 12 ° / polyacrylamide gel electrophoresis (gel thickness: 0.35 dish, gel buffer: 0.112M Tris-0.112M acetic acid (pH6.4), outer tank Buffer solution: 0.025 M Tris-0.088 M glycine (PH8.8) discontinuous buffer system), followed by silver staining.

(3) PCR-RFLP:

For RFLP, the amplified fragment (185 bp) obtained by nested PCR was purified by alcohol precipitation, and then hydrolyzed with SmaI (Toyobo, Osaka, Japan) at 37 ° C for 6 hours. Thereafter, 8% polyacrylamide gel electrophoresis was performed in a pH 8.3 buffer solution of 0.114 M tris-boric acid (containing 2 mM EDTA), and cytosine (C) at position −153 was obtained by staining with bromide chidium. Was searched for the presence or absence of a 163 bp fragment specific to the sequence that was replaced.

(4) ¾ 西?. Ιί ：

Abnormal bands in SSCP were re-amplified under each PCR condition, DΝΑ was extracted and purified using DEAE paper, and the nucleotide sequence was determined using an auto-sequencer (ΑΒΙ373Α). The results of this screening of Japanese patients with open-angle glaucoma are as follows. The intron at the boundary between exon and intron has no abnormality. However, three abnormalities were found in the promoter area. One is the guanine (G) to adenine (A) polymorphism at position -83. These have already been reported by Alward, WL et al. (N. Engl. J. Med. 1998; 338: 1022-1027).

The second abnormality was a new mutation in the GT repeat at positions -339 to -314. These are considered to be statistically polymorphic.

However, as a third abnormality, a new mutation was observed in the promoter mutation from thymine (T) at position 153 to cytosine (C).

This substitution of thymine (T) for cytosine (C) results in a tandem repeat of CAGCCCCAC. Moreover, the CACCC motif, which is known to be involved in the expression of the aglobin gene, connects these two repeats [5 'CAGCCC £ AiUilIiIAGCC (TC) CAC]. Therefore, it was thought that this mutation might affect the expression of the myocillin gene.

This mutation was detected in a very large number of patients, about 20% of open-angle glaucoma, including normotensive glaucoma. This high frequency is a remarkable figure in genetic studies as a single disease-related mutation. Since the history of this discovery was the most important motivation for applying for a patent for the present invention, the details will be described below in accordance with experiments. As shown in the table, in the first survey, this gene was found in 17% of glaucoma, but in the control group, mutation was found in 7.9%. There was no significant difference.

However, in the first survey, three people with cytosine (C) at position -153 (indicated by * in the table below) were examined ophthalmologically and, surprisingly, all three had normal tension glaucoma. Had open-angle glaucoma. In other words, the three healthy individuals, who had no ophthalmologic subjective symptoms, had either advanced glaucoma or early glaucoma. Thus, in practice, the patient group was 8 out of 33 (24.2%) and the healthy person was 0 (0%) of 35. That is, it was strongly suggested that the substitution of thymine (T) to cytosine (C) at position −153 is a single gene abnormality that is very promising for detecting glaucoma. Based on these results, we concluded that the replacement of thymine (T) with cytosine (C) at position -153 was a new target for 53 patients with open-angle glaucoma, including normotensive glaucoma; A second survey was conducted on 63 age-matched healthy individuals.

In the second study, as in the first study, the mutation from thymine (T) to cytosine (C) at position -153 was detected in a large number of patients. (P <0.01, by Fisher's exact probability calculation). Even when the results of the first and second surveys are combined, the differences between patients and healthy individuals are evident (Table). At position -153, thymine (T) was replaced with cytosine (C), but no homozygotes were detected.

According to the table, the combined mutations in the first and second surveys were 16 in the patient group and 6 in the control group. It is not known whether glaucoma or glaucoma is present in the second group of controls since the third group had not performed an ophthalmic examination. However, a total of 19 in the first survey, where a substitution of thymine (T) for cytosine (C) was detected at position -153, and 11 in the patient group in the second survey, a total of 19 The person had normal tension glaucoma or open angle glaucoma. Patients were characterized by the fact that most had a maximum intraocular pressure of 30 mmHg or less.

Aberrant myosin promoter in open-angle glaucoma, including normal tension glaucoma

* ·-These three people belonged to the control group, but actually all of them had glaucoma,

0/38 (0%).

**: Perform ophthalmic examination, none 1

***: Of the 6 controls in this control group, 3 had clear glaucoma, and the remaining 3 tested for glaucoma or non-glaucoma.

-83G → A means substitution of guanine (G) to adenine (A) at position -83, and n in the column of (GT) n represents the number of repeats of GT. -153T → C means substitution of thymine (T) to cytosine (C) at position -153. (GT) n is statistically polymorphic. From the results in the table, the mutation of thymine (T) to cytosine (C) at position -153 is clearly associated with glaucoma. This mutation was also commonly found in the first survey, the second survey and in total, in about 20% of patients with open-angle glaucoma.

In glaucoma patients with thymine (T) to cytosine (C) mutation at position -153, Such a high concordance rate has not been reported for a previously reported mutation associated with juvenile open-angle glaucoma, including all exons, introns, and promoters.

Preliminary studies have shown that promoter activity is clearly different for the thymine (T) and cytosine (C) alleles at position -153, and that the change in promoter activity is due to the GT nucleotide 160 nucleotides upstream of this mutation. It was suggested that it is related to the linkage between the number of dinucleotide repetitions and.

This allele at position -153, a cytosine (C), was first discovered in our study and is the major mutation associated with open-angle glaucoma, including normotensive glaucoma. This gene is significantly more frequent than previous reports of normal tension glaucoma and juvenile open angle glaucoma.

For patients who are worried about inheritance in their offspring and young people who have glaucoma in their family and are at risk of having no carrier, the gene of the present invention can be used for screening when the onset cannot be detected clinically. Is also useful.

The τ ability of the il ffl

ADVANTAGE OF THE INVENTION According to this invention, a glaucoma patient can be detected at an early stage, and the person who is highly likely to suffer from glaucoma can be detected with high accuracy not reported in the conventional gene. In particular, in Japan, genetic diagnosis is possible for the first time for normal-tension glaucoma with a large number of patients and few subjective symptoms. Glaucoma patients, including normal-tension glaucoma, before or in parallel with multiple complex tests that require time and effort, such as measuring intraocular pressure and visual field, based on the gene of the present invention. In addition, groups with a high likelihood of glaucoma in families can be detected. In addition, in order to respond to the genetic concerns of the affected individual, the glaucoma-related gene of the present application can be obtained using the glaucoma diagnosis kit or method of the present invention, if desired, even if the onset cannot be confirmed ophthalmologically. It is also valuable because it can be tested for the presence of a glaucoma and whether the trait associated with glaucoma is inherited.

Claims

Opening claims

1. A polynucleotide comprising the DNA sequence represented by SEQ ID NO: 1.

2. A polynucleotide containing a part or all of a polynucleotide in which thymine (T) at position 153 is substituted with cytosine (C) in the DNA sequence upstream of the human myosillin gene represented by SEQ ID NO: 2. A polynucleotide comprising at least the base at position -153.

3. The polynucleotide according to claim 2, wherein one or several bases are deleted, added or substituted at a position other than the -153 position.

4. The polynucleotide according to claim 2 or 3, wherein the nucleotide length is 19 to 3 16 bp.

5. A primer used for amplifying the polynucleotide according to any one of claims 1 to 4.

6. (i) a primer of SEQ ID NO: 3,

(ii) one or more primers selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6

A pair of primers.

7. A kit for detecting the substitution of thymine (T) to cytosine (C) at position -153 of the promoter region of a human myosin gene, comprising the primer of claim 5 or claim 6.

8. A glaucoma pre-onset diagnostic kit comprising the primer of claim 5 or claim 6.

9. A probe represented by SEQ ID NO: 7 for detecting cytosine (C) at position -153 of the promoter region of the human myosillin gene.

10. A probe represented by SEQ ID NO: 8 for detecting the case where thymine (T) is located at position −153 of the promoter region of the human myocillin gene.

11. Detection of the substitution of thymine (T) to cytosine (C) at position -153 of the promoter region of the human myocillin gene, including the probes represented by SEQ ID NOs: 7 and 8, kit.

12. A glaucoma pre-onset diagnostic kit comprising a probe represented by SEQ ID NO: 7 or SEQ ID NO: 8.

13. The diagnostic kit for glaucoma according to claim 8, wherein the glaucoma is normal tension glaucoma.

1 4. For those who are likely to suffer from glaucoma, which detect the substitution of thymine (T) to cytosine (C) at position -153 in the DNA sequence of the promoter region of the human myosillin gene. Detection method.