JP2002238577A - Cerebral aneurysm-sensitive gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clone a onset risk gene of cerebral aneurysm. SOLUTION: A method for determining the presence of risk of onset of cerebral aneurysm comprises identifying genetic polymorphism of intron part of a human elastin gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene involved in cerebral aneurysm (subarachnoid hemorrhage) and use of the gene.

[0002]

[Prior Art] According to the "Trends in National Hygiene", the mortality rate of cerebrovascular disease (stroke) has been steadily reduced since the 1960s due to thorough management of hypertension and improvement of living environment. Was third. However, although stroke mortality has declined, the total number of patients has increased significantly due to advances in treatment methods and the number of mild cases, which is the largest cause of so-called “bedlessness”. The mortality rate of subarachnoid hemorrhage among strokes has been increasing gradually since 1951. Despite the remarkable progress in surgical therapy, only a subset of patients with subarachnoid hemorrhage will be treated, and about half will die of the first bleed. Especially in the elderly, subarachnoid hemorrhage has many severe cases, poor surgical results, and often leads to bedridden. For this reason, early prevention and secondary prevention of early treatment (detection and treatment before bleeding) are definitely required. However, for early detection and early treatment,
For example, what to index besides managing hypertension
It is still unknown.

[0003] On the other hand, epidemiological studies so far have revealed that the occurrence of cerebral aneurysms has a relatively strong genetic background. With the cooperation of 1,100 neurosurgery-related facilities nationwide, 104 affected sib pairs (branch aneurysm patient siblings) were successfully collected. 85 families were collected, of which 77 were
2 brothers, 7 brothers, 1 brother, 7 brothers
In 3 families, the proband had subarachnoid hemorrhage (ruptured cerebral aneurysm confirmed), and in 12 families, only the unruptured cerebral aneurysm was observed (Kasuya, H., et al., Neurosurgery, 46
(6) 1301-1306 (2000)). It is an indicator of the strength of genetic factors,
The relative risk factor (λs) for siblings is 6.

[0004] Although cerebral aneurysms are thought to be complicated by genetic and environmental factors, leading to the onset of the disease, the genes responsible for cerebral aneurysms are not yet known.

[0005]

SUMMARY OF THE INVENTION The present inventors identified loci by linkage analysis in order to clarify the genes responsible for cerebral aneurysms, and as a result, the loci were highly relevant to the occurrence of cerebral aneurysms. The responsible gene was found.

An object of the present invention is to identify a responsible gene highly associated with a cerebral aneurysm (subarachnoid hemorrhage). By identifying the gene, a human having a genetic risk of a cerebral aneurysm is identified, It is possible to establish a secondary prevention system to diagnose before cerebral hemorrhage occurs.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above problems, and as a result, one of the cerebral aneurysm loci was identified as 7q1 by linkage analysis of siblings suffering from cerebral aneurysms.
The elastin gene was identified as a promising candidate gene identified in the 1.23 region. It has been known that polymorphisms exist in exon 20 (Nucleic Acids Research 19, 4314. 1991) and 3UTR1 (Human Gent., 104, 135-142) of the elastin gene. Again, as a result of examining the association between the elastin gene polymorphism and cerebral aneurysm by haplotype analysis, it was found that the intron portion of the elastin gene, specifically, intron 20 and intron 23 haplotypes are significantly more frequent in patients. It has been found that the genotyping of the combination of intron 20 and intron 23 can be used for early detection and early treatment of cerebral aneurysm, and diagnosis of prevention of subarachnoid hemorrhage can be diagnosed.

That is, according to the present invention, a polymorphism (wild type: T, mutant type: C) of the 17th DNA of the 20th intron DNA sequence of the human elastin gene,
In addition, a gene having a gene polymorphism (wild type: T, mutant type: C) of the 24th DNA of the 23rd intron site DNA sequence is provided, and the polymorphism in intron 20 is a wild type (T) and an intron. A haplotype in which 23 gene polymorphisms are mutated to C is provided.

According to the present invention, DNA obtained from a specimen
A method for determining the presence of a risk of cerebral aneurysm by detecting the haplotype is provided. Specifically, the present invention identifies a polymorphism at the 17th DNA of the 20th intron DNA sequence of the human elastin gene and a 24th DNA polymorphism at the 23rd intron DNA sequence of the human elastin gene. The present invention provides a method for determining the presence of a risk of developing a cerebral aneurysm.

Further according to the present invention, the method comprises direct nucleotide sequencing, allele-specific oligonucleotide (ASO) -dot blot analysis, single-base primer extension, PCR-single-strand conformation. By detecting the presence of said haplotype characterized by using any of polymorphism (SSCP) analysis, PCR-restriction fragment length polymorphism (RFLP) analysis, invader method and quantitative real-time PCR detection method A method is provided for determining the presence of a risk of cerebral aneurysm. Preferably, for example, a single-base primer extension method, specifically, a snapshot (S
There is provided a method for determining the presence of a risk of cerebral aneurysm, characterized by using a NaPshot method or a pyrosequencing method.

Further, according to the present invention, at least human
It is possible to provide an oligo DNA which is a partial sequence of the elastin gene, wherein the 20th intron DNA sequence is a wild type and the 23rd intron DNA sequence is a mutant.

Further, according to the present invention, a gene therapy agent comprising an oligo DNA as an active ingredient for converting the 24th nucleic acid sequence of intron 23 of the human elastin gene to a wild type, and a gene therapy using the gene therapy agent Law can be provided.

Further, according to the present invention, there is provided a method for screening a candidate compound which affects the haplotype of the elastin gene.

The present invention provides the following inventions.

Item 1. A method for determining the presence of a risk of developing a cerebral aneurysm by identifying a genetic polymorphism in an intron portion of a human elastin gene.

Item 2. The polymorphism in the intron portion of the human elastin gene is such that the 17th DNA of the 20th intron site DNA sequence is a wild type (T), and the 24th DNA of the 23rd intron site DNA sequence is Item 2. The method according to Item 1, wherein it is determined that the risk of developing a cerebral aneurysm is high in the case of a haplotype (and a complementary strand thereof) comprising a mutant type in which a wild type (T) is mutated from C to C.

Item 3. Genetic polymorphisms can be determined by direct nucleotide sequencing, allele-specific oligonucleotides (AS
O) -dot blot analysis, single base primer extension, P
Using a method including at least one selected from the group consisting of CR-single-chain higher-order polymorphism (SSCP) analysis, PCR-restriction fragment length polymorphism (RFLP) analysis, invader method and quantitative real-time PCR detection method. Item 3. The method according to Item 1 or 2, wherein

Item 4. Item 4. The method according to Item 3, wherein the identification is performed using a single nucleotide primer extension method.

Item 5. An oligonucleotide comprising the following (a) and (b): (a) an oligonucleotide as a primer or probe for detecting a polymorphism capable of hybridizing to a human elastin gene, wherein the oligonucleotide is human elastin A sequence containing the polymorphic site of the 17th DNA of the 20th intron site DNA sequence of the gene or the 3 ′ end of the oligonucleotide located one to several bases upstream from the polymorphic site of the gene Oligonucleotides, (b) oligonucleotides as primers or probes for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide has a DNA sequence of the 23rd intron site DNA sequence of the human elastin gene. 24th DNA gene Oligonucleotide 3 'end of the sequence or the oligonucleotide comprising a mold portion is to be located a few bases upstream from one base than the gene polymorphism site.

Item 6. A primer for detecting a polymorphism at the 17th DNA of the 20th intron DNA sequence of the human elastin gene, the 24th DN of the 23rd intron DNA sequence of the human elastin gene
A diagnostic kit for detecting the risk of developing a cerebral aneurysm, comprising a primer for detecting the gene polymorphism of A.

Item 7. The 17th DNA of the 20th intron site DNA sequence of the human elastin gene is wild type (T), and the 23rd intron site D
DNA for detecting the risk of developing a cerebral aneurysm, wherein the DNA at position 24 of the NA sequence is a mutant form in which the DNA is mutated from T to C
A haplotype consisting of a sequence (and its complementary strand).

Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in the present specification are referred to as IUPAC.
-IUB regulations [IUPAc-IUB communication on Biologi
calNomenclature, Eur.J. Biochem., 138: 9 (198
4)], "Guidelines for preparation of specifications containing base sequences or amino acid sequences" (edited by the Patent Office) and conventional symbols in the relevant field.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, "gene polymorphism"
The term "is" refers to the presence of two or more types of bases in humans at a certain site in a gene. In this case, the common type is mutated to a wild type for a high-frequency type and to a mutation having a low-frequency type base different from that in a general population. It is called type.

A haplotype refers to a combination of gene mutations present in one allele (haploid).

One specific example of the gene of the present invention is a human elastin gene named intron 20 / intron 23 haplotype shown in Examples described later.
17th DNA of the 0th intron site DNA sequence
Is wild type and the intron site DN at position 23
A haplotype consisting of a mutant in which the 24th DNA of the A sequence is mutated from T to C can be mentioned.

As shown in FIG. 4, the full-length sequence of the human elastin gene was 55 kb in length by Indik et al.
(Bashir, M., et al., J. Biol. Chem., 264, 8887-8).
891 (1989).

The inventor of the present invention found that 104 pairs of siblings with cerebral aneurysms (branch patients with cerebral aneurysms) showed strong genetic factors.
It was expected that the loci could be sufficiently identified by the sibling logarithm by simulation considering (λs = 6), and linkage test was performed by the affected sib-pair linkage analysis method. As a result of linkage analysis over the entire genome, marker D7S2 on chromosome 7
472 had the strongest linkage result. As a result of multipoint linkage analysis, the maximum lod = 3.22 was obtained in this region. That is, one of the cerebral aneurysm loci can be identified as 7q11.23,
It was strongly suggested that a disease gene was present in this region.

The present inventor has found that the strongest linkage was detected at 7q1.
It was found that the elastin gene was present adjacent to the 1.23 region genetic marker, and the gene mutation was screened for the gene by direct sequence analysis (see Example 3). Nucleotide polymorphism was detected. In addition, the present inventors performed a case-control study comparing the association between elastin gene polymorphisms and cerebral aneurysms for all polymorphisms in allele variants, but did not result in significant differences for individual mutations . However, haplotype analysis performed by combining the two polymorphisms revealed that the intron 2 of the human elastin gene was
The haplotype in which the 17th DNA of 0 was mutated to wild-type T (thymine) and the 24th DNA of intron 23 to C (cytosine) obtained the strongest significant difference, and the present inventors combined these two mutations. As a result, they found that they are involved in cerebral aneurysm disease.

In the present invention, the gene is intended to include not only double-stranded DNA but also single-stranded DNA such as a sense strand and an antisense strand which constitute the gene, and is not limited in its length. is not. Therefore, unless otherwise specified, the gene (DNA) of the present invention includes double-stranded DNA including human genomic DNA, and single-stranded DNA.
(Sense strand), single-stranded DNA having a sequence complementary to the sense strand, and fragments thereof.

In the present invention, the gene (DNA) is
Contains regulatory regions, coding regions, exons, and introns.
Examples of the polynucleotide include RNA and DNA, and include genomic DNA and synthetic DNA.

As described above, the provision of the haplotype of the gene of the present invention and the method for detecting the same provide extremely useful information and means for elucidating, grasping, diagnosing, preventing, and treating the risk of cerebral aneurysm. In addition, the haplotype of the gene of the present invention can be suitably used for the development of a novel drug that modifies the haplotype of the gene of the present invention used for the treatment of the cerebral aneurysm into, for example, a wild type. Further, detection of the haplotype of the gene of the present invention in an individual or tissue can be suitably used in elucidation and diagnosis of the cerebral aneurysm.

The synthesis of the gene (or DNA) of the present invention
Chemical synthesis by the phosphoramidite method or triester method can also be used, and it can also be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment synthesizes the complementary strand and allows the strands to anneal together under appropriate conditions, or
Depending on whether the complementary strand is added using NA polymerase,
It can also be obtained from chemically synthesized single-stranded products.

As a specific embodiment of the haplotype of the present invention, for example, the 17th DNA of the 20th intron site DNA sequence shown in SEQ ID NO: 1 of the human elastin gene is T, and An example is a haplotype consisting of a DNA sequence in which the 24th DNA of the 23rd intron site DNA sequence shown in SEQ ID NO: 2 is mutated from T to C.

Further, the haplotype of the present invention is a DNA comprising the 20th intron site DNA of the human elastin gene.
The 17th DNA of the sequence is T, and the 24th DNA of the 23rd intron site DNA sequence is composed of a DNA sequence having a haplotype complementary strand of a DNA sequence mutated from T to C. Include.

The gene containing the haplotype of the present invention may be a full-length gene or a partial gene containing the haplotype.

The gene of the present invention can be prepared based on the sequence information of a specific example of the gene of the present invention disclosed by the present invention.
It can be easily produced and obtained by general genetic engineering techniques [Molecular Cloning 2d Ed, Cold Spring H
arbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.).

Specifically, a genomic DNA library having the haplotype of the gene of the present invention, for example, containing the portions of SEQ ID NOS: 1 and 2 is prepared from an appropriate source according to a conventional method, and It can be carried out by selecting a desired clone using an appropriate probe containing a mutation specific to the invention gene.

In the above description, examples of the origin of the genomic DNA include various cells and tissues having the gene of the present invention, and cultured cells derived therefrom. Specific examples include body fluids such as blood such as serum or plasma, saliva, lymph, airway mucus, urine, and semen.

The method for screening the gene of the present invention from a genomic DNA library is not particularly limited, either, and any conventional method can be used. For example, plaque hybridization, colony hybridization, or the like using a probe that selectively binds to a target DNA sequence, or a combination thereof can be exemplified.

Further, by amplifying the extracted gene by a gene amplification method, screening can be made easier and more accurate. Examples of the gene amplification method include the PCR method (Saiki, RK, Bugawan, TL,
et al., Nature, 324, 163-166 (1986)), NASBA method (Comptom, J., Nature, 650, 91-92 (1991)), TMA method (K
acian, DL, and Fultz, TJ, U.S. Patent No. 5,399,491
(1995)) and the SDA method (Walker, GT, Little, M.
C., et al, Proc. Natl. Acad. Sci. USA, 89, 392-396.
(1992)).

For obtaining the gene haplotype of the present invention, a DNA amplification method by the PCR method [Science, 230, 1350 (1985)] can be suitably used (Saiki, RK, Buga).
wan, TL, et al., Nature, 324, 163-166 (1986)).

The primers used when employing such a gene amplification method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method. In addition, isolation and purification of the amplified DNA fragment can be performed according to a conventional method as described above. For example, it may be performed by gel electrophoresis, or may be purified by a column. Or it can also be observed with a mass spectrum.

The gene haplotype of the present invention obtained as described above can be obtained by a single-base primer extension method as described below, or a DNA fragment thereof can be obtained by a conventional method such as the dideoxy method [Proc. Natl. Acad. Sci., USA ., 74, 5463 (197
7)) and the Maxam-Gilbert method [Methods in Enzymolog
y, 65, 499 (1980)], or simply, using a commercially available sequence kit or the like.

According to the gene (haplotype) of the present invention thus obtained, for example, the presence or absence of the gene of the present invention in an individual or various tissues can be determined by utilizing a part or the entire nucleotide sequence of the gene. Can be detected.

Determination Method of the Present Invention The present invention relates to a method for determining the risk of developing a cerebral aneurysm by identifying a genetic polymorphism of the human elastin gene. Specifically, the method of the present invention is to determine by identifying a gene polymorphism in the intron portion of the gene. Specifically, as described above, 2
The 17th polymorphism in the 0th intron site (hereinafter sometimes referred to as "intron 20") and the 24th polymorphism in the 23rd intron site (hereinafter sometimes referred to as "intron 23"). By identifying the type, the presence of a risk of developing a cerebral aneurysm is determined.

The method of determining the risk of developing a cerebral aneurysm by identifying the two polymorphisms is not particularly limited, as long as the method can identify the genotype of the gene to be detected. For example, the following method may be used.

(1) Nucleic acid containing a gene encoding human elastin (human elastin gene) (D
NA). The DNA may be a full-length DNA of human DNA, or may be a partial DNA containing at least the polymorphism position of the gene of the present invention.

The DNA is not particularly limited as long as it is derived from a human, and can be collected from a biological sample containing human DNA, such as blood or biological material tissue. The method of extracting DNA from these samples may be performed according to a conventional method, for example, using a commercially available kit or device (QIAGEN Blood & Cell Culture DNA kit (QIGEN
AGEN))).

(2) Identification method of the present invention When the amount of DNA obtained in the above (1) is very small, it is amplified by the gene amplification method as described above before the detection method described below. This makes identification easier and more accurate. In the above method and each of the following methods, the DNA fragment to be amplified by the gene amplification method as a specimen is not particularly limited as long as it contains at least one specific site where the presence of the above-mentioned mutation is assumed. In general, it should have a length of about 50 to several thousand bases, preferably 50 to several hundred bases, and particularly preferably contain at least all of the polymorphism sites.

As a probe or primer used in the following detection method, DNA or the like chemically synthesized with a mutation-containing portion based on information on the DNA base sequence of the gene haplotype of the present invention can be used. A DNA fragment containing the haplotype of the gene of the present invention or a mutant base thereof which has already been obtained can also be used favorably. In addition, a primer set based on the nucleotide sequence information of the gene haplotype of the present invention can be used as a screening probe. Or, even without including the gene polymorphism site,
There is no particular limitation as long as it can hybridize to the sense or antisense strand of the human elastin gene and can be used in the following method.

More specifically, the nucleotide sequence used as the probe or primer is such that the 17th nucleic acid of the DNA sequence of intron 20 of the human elastin gene and the 24th nucleic acid of the DNA sequence of intron 23 can be detected. A partial nucleotide sequence corresponding to the sequence set in the above, wherein at least 15 contiguous bases, preferably at least 20 contiguous bases, more preferably 30 contiguous bases, and most preferably 5 contiguous bases.
Those having zero consecutive bases are included, or the positive clone itself having the above sequence can be used as a probe.

1) Nucleotide direct base determination method First of all, detection of the gene according to the present invention is commonly used for determining the nucleotide sequence of this type of gene, for example, the dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci. USA,
74, 5463-5467 (1977)), the Maxam-Gilbert method (Me
thods in Enzymology, 65, 499 (1980)].

Further, the method can be carried out according to a method in which these methods are combined with a DNA amplification method such as the PCR method described above. In particular, from the viewpoint that simple and easy detection with high sensitivity and accuracy can be performed using a small amount of DNA sample, a method combining a PCR method or a DNA amplification method according to the method is preferable.

In this preferred method, most basically, for example, a gene fragment (sample) amplified by PCR is cloned into a plasmid, and then the nucleotide sequence is directly sequenced according to the dideoxy method, the Maxam-Gilbert method or the like. This can be carried out by simply determining the nucleotide sequence using a commercially available sequencing kit or the like. Thus, the presence of a mutation at a particular polymorphic site in introns 20 and 23 of the human elastin gene can be determined and its haplotype determined.

2) Allele-specific oligonucleotide-dot plot analysis As an alternative to the detection method according to the present invention, an allele-specific oligonucleotide (ASO) dot blot method (Conner,
BJ, et al., Proc. Natl. Acad. Sci., 80, 278-282 (1
983)). The method uses, for example, a forward primer and a reverse primer designed to sandwich a single nucleotide polymorphism site of interest, a DNA hybridizing to an allele-specific oligonucleotide probe for a PCR-amplified gene fragment. Fragments can be performed by dot blot analysis. Thus, it is possible to determine whether a single nucleotide polymorphism exists in the DNA fragment.

3) Single-base primer extension method The elastin gene polymorphism (haplotype) of the present invention can be detected by a snapshot method (Kuppuswamy, MN, e).
Natl. Acad. Sci. USA 88, 1143-1147 (19
91)), pyrosequencing (Ronaghi, M., et al., Scie
nce, 281, 363-365 (1998)) and a single nucleotide primer extension method such as the point mutation detection method disclosed in JP-A-2000-279197. In these cases, a primer set to correspond to the base immediately before the target polymorphism site or a base several bases before, that is, the 3 ′ end thereof was set one base upstream or in the vicinity of the mutation to be detected. Primers can be annealed to the DNA sample. Each of the above methods can be carried out using a commercially available kit for detecting SNPs (single nucleotide polymorphism) and software attached to the kit.

For example, in the snapshot method, ABI P
RISM SNaPshot ddNTP Primer
It can be carried out using an Extension Kit (manufactured by ABI Biosystems). The single nucleotide polymorphism is determined by converting the fluorescent fragment generated after the reaction into ABI PRISM310.
/ 337/3100/3700 DNA Analyze
r (both manufactured by ABI Biosystems) and GeneS
Can be detected and analyzed using can software.

The pyrosequence method can be implemented, for example, as follows. That is, genomic DNA is isolated from a blood sample or the like by a conventional method, and tens to hundreds of bases including the polymorphic site are converted to P
CR amplified and single-stranded DNA using magnet beads
And the purified DNA is used as a sample. The sample is annealed with a primer set to sequence from a few bases upstream of the desired mutation, and then dNTPs are added to the device one by one according to the sequence near the mutation input to the software. When the DNA polymerase base-extends, it generates pyrophosphate (PPi).
i was returned to ATP by sulfurylase, and this was used as a substrate for luciferase, a luminescence detector,
Chemiluminescence is detected using a CCD camera or the like. Thus, gene typing becomes possible by analyzing the peak of luminescence obtained in response to the added dNTP. Using this method, 96 samples can be typed in about 15 minutes.

In the above-mentioned method, as the reagents and devices, conventional ones such as DNA polymerase and ATP-
Sulfurylase, luciferase and apyrase (apy
rase), luciferin and APS
Substrate solution composed of (adenosine 5 'sulfate phosphate), dAT
P (deoxyadenosine α-thio-3-phosphate), dCT
A commercially available SNP Reagent Kit (Pyros) comprising dNTP consisting of P, dGTP and dTTP as a constituent element
reagents such as equencing AB) and the PSQ96 system (Pyrosequencing AB) for automatic DNA sequence analysis and SNP software (Pyrose
quencing AB).

In the pyrosequencing method, a nucleic acid is isolated, amplified, the amplified PCR product is purified, and the REA is purified, for example, as described in US Pat. No. 6,159,693.
The reaction can also be carried out by reacting pyrophosphoric acid with DIT ^™ System (manufactured by Promega Corporation) and analyzing the obtained data. For this data analysis, for example, Excel analysis using a commercially available READIT technology (manufactured by Promega Corporation) can be adopted.

4) PCR—Single-Strand Higher-Order Structural Polymorphism (SSCP) Analysis Method Further, in the detection method according to the present invention, the PCR amplification product (single-stranded DNA) described above is subjected to non-denaturing polyacrylamide gel electrophoresis. The PCR-SSCP method (Orita, M., Iwahara,
H., et al., Proc. Natl. Acad. Sci. USA, 86, 2776-2.
770 (1989), Orita, et al., Genomics, 5, 874-879 (198
9)) can also be adopted.

5) PCR-restriction fragment length polymorphism (RFLP)
Analysis method In the detection of a genetic polymorphism or haplotype of the elastin gene of the present invention, when the nucleic acid sequence containing the mutation to be detected contains a restriction enzyme recognition site, the detection is performed by restriction enzyme fragment length polymorphism analysis. Method (RFLP method: Botstein, D.
R., et al., Am. J. Hum.Gen., 32, 314-331 (198
0)).

Specifically, whether the 17th nucleic acid sequence of intron 20 of the elastin gene is a wild type (T) or a mutant type (C), and whether the 17th nucleic acid is a wild type (T)
To determine whether the nucleic acid at position 24 of intron 23 is wild type or mutant, and whether the nucleic acid at position 24 is T or C. Restriction enzymes capable of recognizing sequences before and after each mutation site for recognition may be used.

The enzyme used in the RFLP method may be any of various known restriction enzymes capable of recognizing the sequence before and after each target mutation site.

The RFLP method is more preferably used for PCR-
The RFLP method, that is, a method of amplifying and preparing a sample DNA in advance by a PCR method or a modified method thereof, and then performing the RFLP method on a large amount of prepared and concentrated sample DNA can be used. Thus, the presence or absence of a specific cleavage site can be detected.

The detection of the haplotype of the elastin gene of the present invention by the PCR-RFLP method is more specifically performed, for example, according to the following method. That is, first, DNA of the elastin gene is extracted from a human biological sample, and a DNA fragment in the region containing the gene polymorphism site of intron 20 and / or 23 of the gene is amplified to obtain a large and concentrated sample sample. . Next, the amplified DNA sample is digested with a specific restriction enzyme (that is, an enzyme capable of digesting only either the wild type or the mutant type), and the DNA cleavage mode (the presence or absence of cleavage, the base length of the cleavage fragment, etc.) Is confirmed according to a standard method.

The detection of the genetic polymorphism or haplotype of the elastin gene of the present invention can be performed by the following invaders.
(Invader) method and quantitative real-time PCR detection method (Ta
qMan method).

6) Invader method The following documents can be referred to for implementing the invader method.・ Lyamichev, V., et al., Nat.Bioltechnol., 17 (3) 2
92-296 (1999) and WO9823774 (98/6/4).

This method does not require the amplification of the target DNA in advance to analyze single nucleotide polymorphisms in genomic DNA, and is carried out as follows.

In order to detect whether or not a polymorphism exists at specific sites of introns 20 and 23 of the elastin gene of interest, first, genomic DNA was isolated, and then a 5 ′ flap having a length of 15 to 50 bases was added. Placing a nucleic acid to be detected at the 3 'end of the 5' flap, and a first target probe consisting of an oligonucleotide of 30 to several hundred bases synthesized to be complementary to the target genomic DNA; An invader oligonucleotide probe composed of an oligonucleotide having a length of 15 to several tens bases synthesized so as to be complementary to the target genomic DNA, except that a nucleic acid is arranged at the 3 ′ end, for example, by an automatic synthesizer. . After allowing these probes to react with the isolated genomic DNA in an appropriate reaction solution, an enzyme that recognizes the invader structure containing the mutant nucleic acid and cuts the 5 ′ flap of the first probe is added.

Thus, if the genomic DNA in the sample is
Has the desired nucleic acid for detection, the first target probe in which a nucleic acid complementary to the nucleic acid for detection is arranged at the 3 ′ end of the 5 ′ flap is used. The first reaction to release the 5 'flap with the nucleic acid at the 3' end is completed. If the genomic DNA in the sample does not have the sequence to be detected, cleavage by the restriction enzyme does not occur.

The 5 ′ flap released from the first probe cleaved with the restriction enzyme acts as an invader oligo in a second invasion cleavage reaction using a fluorescence resonance energy transfer (FRET) probe as a target. Continue the reaction sequence.

Thus, the 5 'flap on the first probe is complementary to the FRET probe used in the second reaction, except for the base at the 3' end.

Next, each FRET used in the second reaction
The probe is constructed to contain the same sequence, regardless of the target to be detected, and consist essentially of two elements: (1) 3 'complementary to the product cleaved from the first reaction. Region, (2) forming a duplex to mimic the single-stranded probe, and the targets hybridizing together,
Self-complementary regions where they contain a reporter fluorescent dye and a quencher fluorescent dye.

When the reporter fluorescent dye is bound to the same probe as the quencher fluorescent dye, the fluorescence intensity of the reporter fluorescent dye is suppressed by fluorescence resonance energy transfer, and the reporter fluorescent dye is the same as the quencher fluorescent dye. When the probe is not bound to the probe, the fluorescence intensity is not suppressed. Thus, the free 5 ′ flap from the cleaved first probe oligo is
When hybridized to a FRET probe, it acts as an invader oligo in a second reaction,
Produces an invasion complex that is recognized by restriction enzymes. Thus, restriction enzyme cleavage of the FRET probe separates the two fluorescent dyes and produces a detectable fluorescent signal. In this way, the signal can be amplified from 1 to 1 × 10 ⁶ times by the combination of the first and second reactions in which the standard fluorescence microtiter plate reader can read and detect the product. In the present invention, the presence or absence of a desired wild-type or mutant nucleic acid or a haplotype can also be detected by using an invader assay.

7) Quantitative real-time PCR detection method The genetic polymorphism of the elastin gene of the present invention can be easily detected by a quantitative real-time PCR detection method (TaqMan method).

The method can be carried out as follows.
That is, first, in order to amplify a DNA fragment containing a nucleic acid site for detecting the presence or absence of a target mutation, a forward primer and a reverse primer consisting of about 15 to about 30 bases are prepared. However, both the forward primer and the reverse primer are prepared so as not to include a nucleic acid site for detecting the presence or absence of a target mutation. Then
As a detection fluorescent probe, an oligonucleotide having a base sequence consisting of 15 to 50 bases to which a reporter fluorescent dye and a quencher fluorescent dye are bonded, and a region where the forward primer hybridizes and a region where the probe hybridizes Create a probe that selects a combination that does not overlap with each other.
The probe is prepared so as to have a sequence complementary to an allele-specific sequence for detecting the presence or absence of a target single-base nucleic acid mutation. Using the primers and probes, the desired DN of the elastin gene to be measured in the sample is determined.
PCR is performed using the A fragment region as a template, and the fluorescence from the reaction solution is measured in real time. Thus, the presence or absence of the mutation can be detected.

The invader assay and TaqMan
As a reporter fluorescent dye used in the method, FAM (6
Fluorescein-based fluorescent dyes such as -carboxy-fluorescein) are preferable, and as the quencher fluorescent dye, rhodamine-based fluorescent dyes such as TAMRA (6-carboxy-tetramethyl-rhodamine) are preferable. These fluorescent dyes are known and commercially available real-time detection P
Since it is included in the CR kit, it can be used. The binding positions of the reporter fluorescent dye and the quencher fluorescent dye are not particularly limited, but usually, the reporter fluorescent dye is bound to one end (preferably the 5 'end) of the oligonucleotide portion of the probe, and the quencher fluorescent dye is bound to the other end. . Incidentally, a method of binding a fluorescent dye to an oligonucleotide is known, for example, Noble et al., Nuc.
Res. 12: 3387-3403 (1984) and Iyer et al., J. Am. C.
hem. Soc. 112: 1253-1254 (1990).

Since the real-time detection PCR method itself is publicly known, and an apparatus and a kit therefor are commercially available,
In the present invention, such commercially available devices and kits can also be used. For example, according to the method described in Japanese Patent No. 2825976, or ABI PR manufactured by PE Biosystems, Inc.
It can be performed according to the ISM 7700 sequencing system user manual.

8) Other Detection Methods Detection of SNPs or haplotypes of the elastin gene of the present invention can be further performed by the following methods which are generally known as a method for determining the base sequence of DNA and a method for detecting mutation. It can also be implemented by various methods.

(A) PCR-SSO method using a sequence-specific oligonucleotide; a probe for each mutation is immobilized on a carrier, and a sample (gene amplification product) is hybridized with the probe, and hybridization based on the presence or absence of mismatch is determined. What determines the difference in efficiency.

(B) PCR-SSP for detecting point mutation
Method: Using a sequence-specific primer for gene amplification in which the base corresponding to the point mutation is set at the 3 'end, a significant difference in PCR amplification efficiency occurs depending on whether the 3' end of the primer is complementary or not. The one using.

(C) PCR-DGGE (denaturing agent concentration gradient gel electrophoresis) method: after mixing a mutant DNA fragment and a normal DNA fragment to form a hybrid bond, the concentration of a denaturing agent such as urea or formamide is gradually increased. When electrophoresed in a raised polyacrylamide gel, 1% at the lower concentration of denaturant compared to homoduplex without mismatch.
Dissociates into main chains. Single-stranded DNA has a higher electrophoresis speed than double-stranded DNA.
Base mutations can be detected.

(D) PCR-DGGE / GC clamp method (S
hefield, VC, et al., Proc. Natl. Acad. Sci. USA, 86,
232-236 (1989)); In addition to the PCR-DGGE method, G
This method compensates for the drawbacks of detection when there are multiple base substitutions, deletions, additions and insertions by connecting a region having a high C content to a DNA fragment to be detected for a mutant nucleic acid.
The method particularly requires a step of adding a GC clamp to a DNA fragment to be subjected to mutation detection.

(E) In addition to the PCR-DGGE method, T4
Nucleic acid mismatch detection using endonucleases (US Pat. No. 6,183,958) Recognizing heteroduplex DNA containing single base mismatches such as T4 endonuclease deletions and insertions,
This is a method using the property of cleavage, in which a mixture of a DNA fragment containing a mutation in a test sample and a labeled sequence-specific probe is subjected to PCR amplification, and then the DNA fragment is cleaved at the mutation site by T4 endonuclease. Then, after further reacting the cleaved DNA with another sequence-specific probe, the DNA fragment is again cleaved at the mutation site with T4 endonuclease, and the cleaved product separated by each cleaving is subjected to polyacrylamide gel. By performing electrophoresis and comparing the generated migration patterns, it is possible to detect whether or not two mutations are present in the test sample.

According to the method, not only can the haplotype of the polymorphic gene be determined, but also the paternal haplotype and the maternal haplotype from the DNA sample can be specified.

(F) RNase protection assay (Finkelst
ein, J., et al., Genomics, 7, 167-172 (1990)) (g) In situ RT-PCR (Nucl. Acids Res., 2
1, 3159-3166 (1993)) (h) Southern blotting (Sambrook, J., et al., Mo.
lecular Cloning a Laboratory Manual. Cold Spring H
arbor Laboratory Press: NY. (1989)) (i) Dot hybridization method (Southern, EM
, J. Mol. Biol., 98: 503-517 (1975)), (j) Fluorescence in situ hybridization (FIS
H: Takahashi E., et al., Hum.Genet., 86, 1416 (199
0)) (k) Competitive genomic hybridization (Comp
arative Genomic Hybridization: CGH: Kallioneimi,
A., et al., Science, 258, 818-821 (1992)), (Spectral
karyotyping: SKY: Rowley, JD, et al., Blood, 93, 2
038-2042 (1999)) (l) Method using a yeast artificial chromosome (YAC) vector clone as a probe (Lengauer, C., et al., Cancer Res.,
52, 2590-2596 (1992)).

(M) NASBA method [Nucleic acid sequen
ce-based amplification, Nature, 350, 91-92 (199
1)].

Preferably, a single-base primer extension method, specifically, a snapshot method or a pyrosequencing method can be used.

The primers used when the above-described gene detection method is employed are not particularly limited as long as they are specific to the gene capable of specifically amplifying only the region containing the gene haplotype of the present invention. It can be set appropriately based on the sequence information of the gene of the present invention. Examples of the primer include those preferably having a length of preferably about 15 to 30 nucleotides and having a nucleotide sequence synthesized so as to sandwich the SNP of each haplotype sequence of the gene of the present invention.

Alternatively, for example, the above-mentioned primer having a 3 ′ end 1 to several bases upstream (5 ′ side) in the sense strand or antisense strand from the single nucleotide polymorphism site can be exemplified.

In the present invention, the above detection method may be set so that the gene polymorphic site of intron 20 and the gene polymorphic site of intron 23 can be simultaneously identified,
You may design so that each genetic polymorphism can be identified separately.

As described above, the gene haplotype of the present invention also includes a DNA fragment used as a specific primer and / or a specific probe for detecting the polymorphic gene according to the present invention.

In the present invention, since a desired polymorphic gene to be detected is detected at a risk of developing a cerebral aneurysm, a gene-specific probe for detection is used, for example, the cerebral artery of the present invention. In diagnosing the presence of a risk of aneurysm,
It is preferable to prepare a polymorphism-specific sequence probe for detecting a haplotype in which the polymorphic site of intron 20 of the elastin gene is wild-type and the polymorphic site of intron 23 is a mutant type. Is done.

More specifically, specificity capable of detecting whether the 17th nucleic acid sequence of intron 20 of the elastin gene is a wild type or a mutant type, ie, whether the 17th nucleic acid is T or C Whether the 24th nucleic acid sequence of the probe and intron 23 site is wild type or mutant type,
That is, any specific probe that can detect whether the 24th nucleic acid is T or C may be used.

Kit The identification method of the present invention can be easily carried out by using a reagent kit for detecting haplotypes of intron 20 and intron 23 of the human elastin gene in a sample. The present invention also provides such a kit. Specifically, the present invention provides a primer for detecting a polymorphism at the 17th DNA of the 20th intron site DNA sequence of the human elastin gene, the 23rd intron site DNA of the human elastin gene,
A diagnostic kit for detecting the risk of developing a cerebral aneurysm, comprising: a primer for detecting a gene polymorphism of the 24th DNA in the sequence.

The primers can hybridize to the sense strand or antisense strand of the above elastin gene, and have the intron 20 and the intron 23.
Primers capable of amplifying each gene polymorphism site, one base to several bases upstream of the gene polymorphism site (5 'side)
And a primer having a sequence having a 3 ′ end, a primer having a sequence having a gene polymorphism site, and the like.

Further, there is provided a reagent kit for detecting the risk of developing a human cerebral aneurysm, which comprises a restriction enzyme that recognizes a nucleic acid sequence of several bases including a polymorphic site.

The reagent kit also comprises at least the intron 20 site and the intron 23 of the elastin gene.
If a DNA fragment that hybridizes to part or all of the DNA base sequence of the site or its complementary base sequence is included as an essential component, a labeling agent,
Reagents essential for the PCR method (eg, Taq DNA polymerase, deoxynucleotide triphosphate, primers, etc.) may be included.

Examples of the labeling agent include a radioisotope and a chemically modified substance such as a fluorescent substance.
The fragment itself may be conjugated with the labeling agent in advance. Further, the reagent kit may contain a suitable reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., for the benefit of performing the measurement.

The present invention also provides a method for diagnosing the risk of developing a human cerebral aneurysm using the above-mentioned measuring method, a diagnostic agent and a diagnostic kit used in the method.

Gene Therapy of the Present Invention The present invention also relates to the intron 2 of the human elastin gene.
3. An oligo DNA for converting the 24th nucleic acid sequence to a wild type, or a gene therapy vector containing the oligo DNA and the vector, the 24th nucleic acid sequence of intron 23 of the human elastin gene being converted to a wild type. To provide a drug containing, as an active ingredient, a cell into which an oligo DNA has been introduced.

That is, according to the present invention, it is composed of several tens to several hundreds of bases, composed of a partial sequence of the human elastin gene,
Oligonucleotide synthesized so that the 24th nucleic acid sequence of intron 23 is T, vector for gene therapy introduction containing the oligonucleotide, cells transfected with the oligonucleotide by the vector, and gene therapy transfection A gene therapy agent comprising, as an active ingredient, a vector for use or a cell into which a wild-type nucleic acid of intron 23 of the human elastin gene has been introduced using the vector.

The oligonucleotide, the gene therapy vector containing the oligonucleotide, the cell into which the oligonucleotide or the vector has been introduced, and the wild type of the human elastin gene intron 23 by the gene therapy introduction vector or the vector. A therapeutic agent comprising a cell into which a type nucleic acid has been introduced as an active ingredient is a mutant type in which the 17th nucleic acid of intron 20 of the elastin gene is a wild type and the 24th nucleic acid of intron 23 is mutated to C. A person at risk for a cerebral aneurysm having a certain haplotype, or a cerebral aneurysm having the haplotype
(Subarachnoid hemorrhage) By administering to the patient's cerebral vascular cells or the patient's peripheral blood tissue site, to improve gene mutations in these tissues, to prevent secondary development of cerebral aneurysm,
Alternatively, a prophylactic and therapeutic agent for cerebral aneurysms characterized by suppressing recurrence in patients with cerebral aneurysms (subarachnoid hemorrhage) can be provided.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. For example, Maniatis, T., et al., Molecular Cloning: a Labor
atory Manual (Cold Spring Harbor Laboratory, cold
Spring Harbor, New York (1982)), Sambrook
rook, J., et al., Molecular Cloning a Laboratory Man
ual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York (1981)), Ausber (ausbe
l, FM, et al., Current Protocols in Molecular Bio
logy, John Wiley and Sons, New York, New York, (199
2)), Glover (Glover, D., DNA cloning, I and II
(Oxford Press) (1985)), Anand, Techniques f
or the analysis of complex genomes, (Academic Press
(1992)), Guthrie (g., Et al., Guide to Yea
st Genetics and Molecular Biology, (Academic Pres
s) (1991)) and Fink (Fink, et al., Hum.
r., 3, 11-19 (1992).

I) Gene analysis for detecting the elastin gene haplotype of the present invention can be performed by the above-described determination method. For example, the intron 20 site of the elastin gene is a wild type and the intron 23 is a mutant type. To detect haplotypes, genomic DNA obtained from cells from human blood or tissue cells was ligated with intron 20 of elastin gene and intron 2
Primers for PCR amplification of genomic DNA are respectively prepared so as to contain each of the 24th nucleic acid sequence of No. 3, and the DNA is amplified and screened to confirm the presence or absence of the human elastin gene haplotype.

These diagnoses can be determined by examining DNA obtained from any human tissue. A preferred method is to take blood and remove D from blood cells.
Extract NA or extract DNA from cells obtained directly from target tissue cells.

Ii) Next, a human having the haplotype in which the gene polymorphism site of intron 20 is wild-type and the gene polymorphism site of intron 23 has a mutant type is referred to as a gene polymorphism of intron 23. A virus or plasmid vector containing a copy of a DNA in which the polymorphic site of intron 23 of the human elastin gene linked to an expression control element capable of converting the site to wild-type is wild-type and capable of replicating in the cell. create. Suitable vectors here are described in US Pat. No. 5,252,479.
And the vectors disclosed in PCT Publication No. WO 93/07282. Next, the prepared vector containing DNA in which the gene site of intron 23 of the human elastin gene is wild type is systemically injected into a patient such as blood and injected into a patient. If the transformed gene is not permanently integrated into the chromosome of the targeted vascular cell, the process can be repeated periodically. As described above, the gene therapy method includes an in vivo method in which a material for gene transfer is directly administered into the body as described above, and a method in which a target cell is once taken out of a patient's body, a gene is introduced outside the body, and then the cell is introduced into the body. Both methods of the ex vivo method of returning can be appropriately selected.

In the method for producing the vector containing the gene of the present invention, the DNA in which the intron 23 of the elastin gene to be introduced is wild-type can be easily prepared by general genetic engineering techniques based on the nucleotide sequence information of the gene. (Molecular Cloning 2d Ed,
Cold Spring Harbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II and III", edited by The Biochemical Society of Japan (1986)). The synthesis of DNA is also as described above.

According to the present invention, a biological sample such as blood or serum is prepared, nucleic acids are extracted, if desired, and analyzed for the presence of the haplotype-sensitive genes of intron 20 and intron 23 of the human human elastin gene. It is possible.

Further, according to the present invention, the presence of a risk of developing cerebral aneurysm can be detected by analyzing the haplotypes of elastin gene intron 20 and intron 23 from a sample such as blood or serum.

[0112]

According to the present invention, a gene haplotype associated with the risk of developing a human cerebral aneurysm is provided.

The gene haplotype of the present invention is strongly linked to the development of human cerebral aneurysms, and is considered to be a factor that promotes the development of cerebral artery blood vessels, particularly subarachnoid hemorrhage, in these cells and their surrounding tissues. The analysis of the polymorphism at the specific site of intron 20 and the polymorphism at the specific site of intron 23 of the human elastin gene of the present invention can be used for studying the relationship between the function of the gene and the onset of cerebral aneurysm. Or to genetic diagnosis for patients with cerebral aneurysms (subarachnoid hemorrhage), secondary prevention, prediction of recurrence of cerebral aneurysms (subarachnoid hemorrhage), indication for surgery, and medical use by wild-type DNA strand against the mutant gene It can be used for applied research.

According to the present invention, there is further provided a gene transfer vector useful for gene therapy containing a wild-type oligonucleotide at the intron 23 site of the human elastin gene, and a wild-type oligonucleotide at the intron 23 site of the human elastin gene. Cell and a gene therapy agent comprising the vector or the cell as an active ingredient, and a gene therapy method utilizing the same.

Further, according to the present invention, the onset of cerebral aneurysm (subarachnoid hemorrhage) caused by the haplotype of the gene polymorphism at the specific site of intron 20 of the human elastin gene and the polymorphism at the specific site of intron 23 of the human elastin gene In addition, a screening method and a screening kit for candidate compounds that reduce the risk can be provided.

[0116]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Genome- Wide Linkage Analysis Materials and Methods : With the cooperation of 1,100 brain surgery-related facilities nationwide, 104 affected sib-pairs (branch patients with cerebral aneurysms)
Collected. The breakdown consisted of 85 families, 77 pairs of 2 brothers, 7 pairs of 3 brothers and 1 pair of 4 brothers. In 73 families, the proband had subarachnoid hemorrhage. In 12 families, the proband was:
Only unruptured cerebral aneurysms were observed (Kasuya, H., et al., Ne
urosurgery, 46 (6) 1301-1306 (2000)). Simulations in consideration of the strength of genetic factors and the risk factor for siblings (λs = 6) were expected to allow sufficient identification of the orphan locus by this sibling logarithm.

That is, when the principle of the method is abbreviated, since the affected sibling has inherited the allele that causes the disease from the parent, the allele must be shared. On the other hand, the number of alleles shared by siblings is 1 (value based on the null hypothesis). By examining the number of alleles shared by many affected sib pairs, when more allele sharing is observed than the number of alleles based on the null hypothesis, "linkage was recognized".

For the purpose of linkage analysis covering the entire genome, 40
Linkage mapping set consisting of 0 fluorescently labeled microsatellite markers
et version II. PE Applied Biosystems). 8.6 on average with this marker set
Analyzes can be performed at centiMorgan intervals. However, since markers with low heterozygosity were present in Japanese, the number of markers was further increased, and analysis of the entire genome was performed using 420 markers.

By using primers labeled with three types of 6-FAM (blue), HEX (green), and NED (yellow) fluorescent dyes, a dinucleotide repeat microsatellite (dinucleotide repeat / microsatellite) ) Could be amplified by PCR.

The size of the PCR product thus obtained was determined by using an automatic analyzer ABI PRISM: Model 377
(Applied Biosystems Co., Ltd.). Since the PCR reaction is carried out using the primers labeled with the three types of fluorescent dyes, the three types of colors can be separately distinguished even for PCR products of the same size. For the 96 samples in the reaction gel, 15
Since the types of markers can be analyzed simultaneously, about 1500 genotyping could be processed by one electrophoresis.

Typing of large samples can be done by using Gene
Rapid processing was performed using a Scan, GenoTyper program (manufactured by PE Applied).

The linkage test was performed using a nonparametric analysis, GENEHUNTER (KruglyaK, L. et al. Am.
J. Genet., 58, 1347-1363 (1996)), and SIBPA
L program (SIBPAL: SAGE, Statistical Analy
sis for Genetic Epidemiology, release 3.1. Depart
ment of Biometry and Genetics. LSU Medical Cente
r, New Orleans, LA).

Analysis results: The determination of the region where linkage is observed was determined by the following Lander and Kruglyak guidelines (Nature Genet, 11, 241 (1)
995)), based on the criteria for obtaining false positive linkages.
Furthermore, since there were many false positives in the linkage analysis of the entire genome, a region where two or more markers with a significant difference P <0.05 were continuously detected was determined to have linkage.

Lander and Cragglyak guidelines: Genome-wide linkage analysis has been actively performed in multifactorial diseases, but it has been determined whether linkage analysis of individual genes can be a cause based on their gene functions. Join. However, in the analysis of the whole genome, gene function is not considered at that stage,
A purely mathematical and genetic significance criterion (threshold) is required. So they follow the simulation and make significant linkage criteria.・ Suggestive linkage: P <7.4 × 10 ⁻⁴ , lod> 2.2, criteria for obtaining one false positive linkage result over the entire genome ・ Significant linkage: P <2.2 × 10 ⁻⁵ , lod > 3.6, criteria for obtaining 0.05 false-positive linkage results across the genome. High Significant linkage: P <3.0 × 10 ⁻⁷ , lod> 5.4, 0.01 across the genome. Criteria for obtaining false positive linkage results of
Does not include the X chromosome. ) To 5 (D5S42
8, D5S644), chromosome 7 (D7S669, D7S630), chromosome 14 (D14S2
58, D14S74). 5th (Chromosome 5), 7
Table 1 shows the results for the chromosomes No. 7 (Chromosome 7) and No. 14 (Chromosome 14).

[0126]

[Table 1]

Subsequently, multipoint linkage analysis using GENEHUTER was performed to further increase the number of markers in the three regions where linkage was recognized, to narrow the linkage region, and to ensure the linkage.

The results are shown in FIGS. As a result, as shown in FIG.
24, maximum lod at chromosome 7 as shown in FIG.
3.22, as shown in FIG.
od 2.31 was obtained. As a result of detailed mapping, P = 0.000027 for marker D7S2472 on chromosome 7
And got the strongest chain result.

The values thus obtained satisfied the Suggestive Linkage of Lander and Cragglac et al. Therefore, one of the cerebral aneurysm loci could be identified on chromosome 7q11.23, strongly suggesting that a disease gene exists in this region.

Example 2 Search for candidate genes for cerebral aneurysms 7q1 showing the strongest linkage obtained in Example 1 above
1.23 region genetic markers, D7S2472, D7S
Examination of candidate genes adjacent to 2415 revealed that the elastin gene was present (http: //www.ncb
i.nlm.nih.gov/genemap99/).

Elastin is the core of elastin fibrils, a major component of the vascular extracellular matrix. Elastin is a molecule responsible for the elasticity of blood vessels and is thought to be involved in strengthening blood vessels (see Dictionary of Molecular and Cellular Biology (Tokyo Kagaku Dojinsha, 1997)). The most common site of cerebral aneurysm is the cerebral vascular bifurcation such as the virus ring, and the relationship between the fragility of the bifurcation and the occurrence of cerebral aneurysm has been pointed out (Carmichael, R., J. Path. Bact.
vol 62, 1-18 (1964)). Based on the results of linkage analysis and the known functional role of elastin, the elastin gene was considered to be a potential disease candidate gene for cerebral aneurysms.

Example 3 Elastin Gene Structure and Screening The elastin gene is located at 7q11.23, as described above, and is located at a length of 86.5 centiMorgans (cM) from the short arm telomere. Exons (Peoples, R., et al. Am. J. Hu
m. Genet. 66 (1) 47-68 (2000)).

For all exons, promoter regions, and some introns of this elastin gene, gene mutations were screened by direct sequence analysis (Rosenblum, BB, et al. Nucleic Ac
ids Res. 25, 4500-4504 (1997)).

Table 2 shows the results.

[0135]

[Table 2]

As can be seen from Table 2, polymorphic mutations were detected at 13 sites. Among the mutations, the mutations that cause amino acid substitution are exon 5 (C → T: Ala → Val at position 61) and exon 20 (11
Fourth G → A: Gly → Ser). Other mutations were present in the intron and promoter sites. For gene mutation typing, the SNaPshot method using a single base extension reaction was used to detect the allele frequency of each mutation using ABI377 DNA sequencer (manufactured by PE Applied Biosystems). The method comprises ABI
The procedure was performed according to the instruction manual of the 377 DNA Analyzer.

Example 4 Case Control Study In order to examine the association between the elastin gene polymorphism and cerebral aneurysm, allele frequencies of all the polymorphisms obtained in Example 3 were compared by a case control study.

The patients studied were angiography,
MRA (magnetic resonance angiography) or 3D CT
A (3 dimentional computed tomographic angiography)
In any of the above examinations, a cerebral aneurysm with a diameter of 4 mm or more was recognized, and a cerebral aneurysm was not observed by the same method as a non-patient (control). 168 patients, 188 non-patients
For each of the names, a comparison of allele frequencies between patients and controls for each polymorphism was made by the chi-test (χ-test).

That is, if a certain gene mutation causes a disease, a difference in the allele frequency between the patient and the control is expected. A case-control study was performed for all polymorphisms, but none of the polymorphisms showed a difference in frequency. Therefore, haplotype analysis was performed by further combining two polymorphisms, and the frequency was compared.
For haplotypes, use the ARLEQUIN program ( http:
//anthropologie.unige.ch/arlequin )
All possible combinations of two locations were analyzed. Table 3 shows the results.

[0140]

[Table 3]

As a result, there was no significant difference with the single mutation, but a significant difference could be obtained by combining the two sites. The haplotype of intron 20 (+17) and intron 23 (+24) obtained the strongest significant difference, and the value was P = 0.000000005 (Table 3). That is, the intron 20 and intron 23 polymorphisms
Although it was not involved alone in the disease, it was found that the combination of the two was involved in the disease.

Therefore, those having these intron 20 and intron 23 polymorphisms have a high risk as a human or patient having a risk of developing a cerebral aneurysm. That is, it can be seen that humans who are 17th wild-type T of intron 20 of elastin gene and have both the haplotypes in which the 24th T of intron 23 is mutated to C have a high risk of developing a cerebral aneurysm.

Example 5 Method for Detecting Risk of Onset of Cerebral Aneurysm Elastin gene intron 20 and intron 2
Among the three haplotype patients, WV (intron 20 was wild type (W) and intron 23 was mutant type (V)) was significantly higher in frequency (this is referred to as WV haplotype).

Looking at the WV haplotype, the relative risk ratio (odds ratio) is 1.91, ie, a person with this haplotype is 1.9 times more cerebral aneurysm than a person without this haplotype. Susceptible to the disease.

In addition, humans having a homozygous WV-type haplotype had significantly more cerebral aneurysms (10.7%
vs. 2.7%, P = 0.002), and the relative risk was 4.39.
That is, it is suggested that individuals having the homozygous WV type have a very high risk of developing cerebral aneurysms. This is generally calculated as follows:

[0146] These results suggest that human elastin gene typing has diagnostic significance for the risk of developing cerebral aneurysms.

Further, since this genotype did not increase in patients with ruptured cerebral aneurysms (subarachnoid hemorrhage), it was expected that this genotype would be involved not in rupture of cerebral aneurysms but in cerebral aneurysm development itself.

Therefore, in order to detect the presence of a risk of developing a cerebral aneurysm, two locations, intron 20 (17th T → C) and intron 23 (24th T → C), were set as the respective measurement targets. You need to genotype it. It is necessary to construct a haplotype from these two locations and detect a haplotype in which the 17th DNA of intron 20 is wild-type T and the 24th T of intron 23 is C.

As the method, a snapshot (SNaPshot method :) for performing a single base extension reaction is preferably exemplified. The method comprises the steps of ABI PRISM SNaP
shot ddNTP Primer Extenti
It can be easily measured with on Kit (manufactured by ABI Biosystems). The fluorescent fragment generated after the reaction was also ABIa PRISM 310/377/3100/37.
00 and GeneScan software.

That is, after genomic DNA is extracted from a specimen obtained from a subject, several tens to several tens of primers are prepared by using primer pairs prepared so as to contain the DNA fragments of the intron 20 and the intron 23 of the human elastin gene. The DNA fragment length of 100 bases is amplified by PCR. This P
Using the CR product as a template, the corresponding primer is annealed immediately before the target mutant DNA, and only one base is extended with a polymerase. For example, in the case of intron 20, when hybridizing to the antisense strand, a wild-type sequence having an arbitrary length, for example, about 16 to 24 nucleotides in length, from the 16th DNA sequence of the intron 20 to the 5 'side of the sense strand. A primer consisting of When hybridizing to the sense strand, a primer consisting of a wild-type sequence having an arbitrary length, for example, about 16 to 24 bases in length, from the DNA sequence No. 18 at the intron 20 site to the 5 ′ side of the antisense strand, such as intron 20 2 complementary to the sequence of Nos. 18 to 37 in the DNA sequence of
Primer consisting of a nucleic acid sequence of 0 base length (SEQ ID NO: 3)
Are synthesized. And ATGC such as 6-FAM and HEX
DdNTPs labeled with different colors of fluorescence
Mix. Since ddNTP is a base whose extension is stopped by extension of one base, a primer extended by only one base is colored depending on the type of the base at the extended end as a result of this reaction. That is, DNA of intron 20 site
It can be determined whether the 17th sequence is a wild type or a mutant type. Similarly, it can be determined whether the 24th DNA in the 23 intron region is a wild type or a mutant type. For example, in the DNA sequence of intron 23 (SEQ ID NO: 2),
A primer consisting of a nucleic acid sequence having a length of 20 bases complementary to the sequence of No. 44 (SEQ ID NO: 4) is synthesized and discriminated as described above.

The primer labeled with ddNTP is separated by electrophoresis, and the fluorescent signal is analyzed and detected. In addition, instead of the method of detecting a fluorescent signal by electrophoresis, a method of detecting a difference in molecular weight accompanying extension of one base by a mass spectrum can also be applied.

As described above, bases incorporated by mutation are different, and are recognized as different fluorescent signals, so that genotyping is possible. Thus, 196 samples
(5 ng DNA by PCR amplification) can be analyzed on one gel (2 hours electrophoresis).

The intron 23 can be similarly detected.

Example 5-2 Method for Detecting Risk of Onset of Cerebral Aneurysm Pyrosequencing (Ronaghi, M., et a
l, Science, 281, 363-365 (1998)).
That is, after a genome is isolated from a blood sample or the like by a conventional method, tens to hundreds of bases of a target containing a mutation are subjected to PCR amplification using biotin-labeled primers. Purify the single-stranded DNA using magnet beads,
Anneal primers set to sequence from several bases upstream of the mutation. Next, the device adds dNTPs one by one according to the sequence near the mutation input to the software. When DNA polymerase base-extends, it produces PPi (pyrophosphate). Pyrophosphate (PPi) is converted to AT by sulfurylase.
It is returned to P, which is used as a luciferase substrate, and chemiluminescence is detected with a luminescence detector or CCD camera. Since a peak of light emission is obtained according to the added dNTP, that is, the base sequence, genotyping becomes possible.

With this method, 96 samples could be typed in about 15 minutes.

The above method comprises the steps of: DNA polymerase, ATP
-Sulfurylase, luciferase, apyrase (apyra
se), a substrate solution comprising luciferin, APS (adenosine 5 'sulfate phosphate), dATP (deoxyadenosine α-thio-3 triphosphate), dCTP, d
Commercially available SNP Reagent Kits (Pyrosequencing A) comprising dNTPs consisting of GTP and dTTP as constituent elements
B), and a PSQ96 system (Pyrosequencing AB) for automatic DNA sequence analysis using the method and SNP software (Pyrosequencin) for its use.
g AB)).

As described above, the human elastin gene intron 20 and intron 23 having the WV haplotype found by the present invention have a high risk of developing cerebral aneurysms. It is possible to detect the risk of developing a cerebral aneurysm.

According to the method for detecting the risk of developing a cerebral aneurysm according to the present invention, a system for secondary prevention is established in which a human having a genetic risk of a cerebral aneurysm is identified and diagnosed before bleeding. Becomes possible. Further, according to the use of the present invention, the present invention can be used for extraction of an appropriate subject for a brain dock or surgery.

Further, by analyzing how the causative gene is involved in the onset of cerebral artery flow disease, it becomes possible to elucidate the causes of cerebral aneurysms, and to develop new cerebral aneurysms based on pathophysiology.
It can be used to develop drugs for (subarachnoid hemorrhage).

[0160]

[Sequence List] SEQUENCE LISTING <110> OTSUKA PHARMACEUTICAL CO., LTD. <120> AN INTRACRANIAL ANEURYSM SENSITIVE GENE <130> 2D01JP <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 < 211> 236 <212> DNA <213> Homo sapiens <220> <221> intron <222> (1) .. (236) <223> INTRON 20 REGION (14289-14524) OF ELASTIN GENE <400> 1 gtgagcctta gtcacatctg gggacatggg ttgagaaggg atgggggctt cttgtctgct 60 cggctctgca ggggcagtgg ggactgtaga tcgggcttga atgtgctcag ggaggagttg 120 ggggagaaga agggaggtcg tatccatgcc ttacagggca gaagagcttt aaacacggct 180 cggaggagac ccaggcacgg cttctgaggg tctctttctt tctcgtttcc ttgtag 236 <210> 2 <211> 2177 <212> DNA <213> Homo sapiens <220> <221> intron <222> (1) .. (2177) <223> INTRON 23 REGION (15550-17726) OF ELASTIN GENE <400> 2 gtaagtcccc ctcacccccg ccattggctc acggagaact gctttctcct gtgccctgtt 180 ctggggtgcc ttggtcctg tggtcctg tggtcctg tggtcctg tggtcctg tgtttctgat taggggagca gggtgagcag tgtga gcctc cctgttccta aagcccctgg 240 tgcctcccag gctattgggg acctgacctc atgctgagct ccagctcccc ttgagggact 300 ccagttctcc cccttctcct tctctttctc cttctccttc ttcttcttgt gctcctcttc 360 cttcttcttc ttcttctttc ttctcctcct cctccttctc cttctccctc ctcctccttc 420 tcattcttct tcttctcctt cctcttcttc tcctcctgct tcttttcctt ctcctccctc 480 ttcctcctcc tcctgcttct cctttcttct ccttcttctt ctttcttctt cttcttcctc 540 tttttctcct tctttcttct tctttctcct tcttcttctt cttcctctgc tccttcttct 600 ttctcctcct tcttctcttc tcttccgtct ttcttctcct ccttttcctc cttcttcttt 660 cttctccttc tccttttttc ttgagatagg gtctagctct gtcacccagg atggggtaca 720 gtgccacaat catagctcac tacagcctca acctcccagt ctcaagcagt ctgcctgctt 780 ccgcccccca agagctgaga ccacaggtgc ccaccaccat gcctggctaa ttttttaatt 840 tttttgtagc gacagcggtc tcactatgtt gctcaggctg gtctcaaact cctaggctaa 900 agcgatcctc ctgcctctgc ctcccaaagt cctgggatta caggcgtgag ccaccacacc 960 cggcctgcag tacttcttgt tccccatctc ttgctacatt tgagggccac cctggcagcc 1020 ccaggtgccc acacttttct gaacatggca aatcgtggca gcaccaattg ta gagctcaa 1080 ctgtatgtca ggccctgggc atggggtctg taggccttgg ccctagggac ctgtgggctg 1140 aaaggttcag atcagaatct ctaggactga ataggccaga gagcatttcg actgcaggtc 1200 tgctgagccc catattctca cacacagcaa tctttattat ttatttattt gagacggggt 1260 ctcacattgt cgcccaggtt ggagtgcagt gatgccgtca gctcgccgca ggcctcaaac 1320 tcctagctta agccattatt cccccttagt atccctagta gctggggcta cagtcacatg 1380 ccaccatgcc cagtttaaaa aaaaaaaaaa ttgtatgcga tcctcccaca ttggcctctc 1440 aaagtgctgg gatgacaggc atgagccaac gtgcctggcc tacaaaaatc ttacagagtt 1500 gattttattt ttcccatttt acagatgtgg aaactgaggt tcccagagct taagtaactt 1560 gcctacagtt gcacagctaa atggtggctg agctgagatt tgaacccaaa gcctttctgt 1620 cttacaaagt cccttatata atgtaaatct gcctccatca gcctcaaatc tccaaggggt 1680 ccttgtcact gaaaaggtta agaactcctg gccaaatgca gcagctcaca actataatcc 1740 cagaactttg ggaggccaag tcgggtggat cacccaaggt caggagttta agaccagcct 1800 ggccaacatg gtgaaaccct gtctctacta aaaatacaaa aaaattagcc gggcatggtg 1860 gtgcgcacct gtagtcccag ctactcagga ggctgaggca ggagactcac ttgaactc gg 1920 gaggtggtgg ttgcagtgag tcgagatcac gccattgcac tccagcctgg gcgatagagt 1980 gagactctgt ctccaaaaaa acaaagttat gaactcctga gcctgcacac acttcatatt 2040 agggaggagg aagctgaggc ccagcaaggg aaagtaactg atccagggtc acacagcaaa 2100 tctatgccag ggccgaggct ccagccctct ttccataagc ttctgtcctc tttgatcagg 2160 tcttggttaa tgatcag 2177 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 ttctcaaccc atgtccccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400 > 4 aagcagttct ccgtgagcca 20

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a drawing showing markers and maximum lods of chromosome 5 by multipoint linkage analysis.

FIG. 2 is a drawing showing markers and maximum lods of chromosome 7 by multipoint linkage analysis.

FIG. 3 is a drawing showing markers and maximum lods by multipoint linkage analysis on chromosome 14.

FIG. 4 shows the structure of the elastin gene (ELN). P
M indicates a promoter, EX indicates an exon, and INT indicates an intron.

Claims (7)

[Claims] 1. A method for determining the presence of a risk of developing a cerebral aneurysm by identifying a genetic polymorphism in an intron portion of a human elastin gene. 2. The intron portion DNA of the human elastin gene has a 20th intron site DNA.
The 17th DNA in the sequence is wild type (T) and the 24th DN in the 23rd intron site DNA sequence
The method according to claim 1, wherein the risk of onset of cerebral aneurysm is determined to be high when A is a haplotype (and its complementary strand) consisting of a mutant type in which a wild type (T) is mutated from C to C. 3. Gene polymorphism is analyzed by direct nucleotide sequencing, allele-specific oligonucleotide (ASO) -dot blot analysis, single-base primer extension, PCR-
Identification using a method including at least one selected from the group consisting of single-chain conformational polymorphism (SSCP) analysis, PCR-restriction fragment length polymorphism (RFLP) analysis, invader method and quantitative real-time PCR detection method The method according to claim 1 or 2, wherein 4. The method according to claim 3, wherein the identification is performed using a single-base primer extension method. 5. An oligonucleotide comprising the following (a) and (b): (a) an oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to a human elastin gene, The nucleotide is a sequence containing a polymorphic site at the 17th DNA of the DNA sequence at the 20th intron of the human elastin gene or the 3 ′ end of the oligonucleotide is one to several bases upstream from the polymorphic site. (B) an oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is the 23rd position of the human elastin gene; 24th D in the intron site DNA sequence Oligonucleotide 3 'end of the sequence or the oligonucleotide containing a gene polymorphism site of A is to be located a few bases upstream from one base than the gene polymorphism site. 6. A primer for detecting a polymorphism at the 17th DNA of the DNA sequence at the 20th intron of the human elastin gene, and a gene at the 24th DNA of the DNA sequence at the 23rd intron of the human elastin gene. A diagnostic kit for detecting the risk of developing a cerebral aneurysm, comprising a primer for detecting a type. 7. The 17th DNA of the 20th intron DNA sequence of the human elastin gene is a wild type (T) and the 23rd intron DNA
A haplotype consisting of a DNA sequence for detecting the risk of developing a cerebral aneurysm (and a complementary strand thereof), wherein the 24th DNA of the sequence is a mutant form in which a T to C mutation has occurred.