WO2002066639A1 - Brain aneurysm-sensitive gene - Google Patents
Brain aneurysm-sensitive gene Download PDFInfo
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- WO2002066639A1 WO2002066639A1 PCT/JP2002/000823 JP0200823W WO02066639A1 WO 2002066639 A1 WO2002066639 A1 WO 2002066639A1 JP 0200823 W JP0200823 W JP 0200823W WO 02066639 A1 WO02066639 A1 WO 02066639A1
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- gene
- intron
- dna
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Definitions
- the present invention relates to a gene involved in cerebral aneurysm (subarachnoid hemorrhage) and use of the gene.
- Cerebral aneurysms may be complicated by genetic and environmental factors However, the genes responsible for cerebral aneurysms are still unknown. Disclosure 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, found a responsible gene highly relevant to the occurrence of cerebral aneurysms.
- An object of the present invention is to identify a responsible gene highly associated with cerebral aneurysm (subarachnoid hemorrhage). By identifying the gene, it is possible to identify a person who has a genetic risk of cerebral aneurysm and establish a secondary prevention system for diagnosing cerebral hemorrhage before it occurs.
- the present inventors conducted intensive research with the main aim of solving the above problems, and as a result, identified one of the cerebral aneurysm loci in the region of 7q1.1.23 by linkage analysis of siblings with cerebral aneurysm Then, the elastin gene was found as a promising candidate gene. It was already known that the gene polymorphism exists in exon 20 (Nucleic Acids Research 19, 4314, 1991) and 3UTR 1 (Human Gent., 104, 135-142, 1999) of the elastin gene. Conducted further studies and examined the association between the genetic polymorphism of the elastin gene and cerebral aneurysm by eight-prototype analysis.
- intron portion of the elastin gene specifically, the intron 20 and intron 23 haplotypes were significantly higher in patients, and the genotyping of the combination of intron 20 and intron 23 showed that It has been found that it is possible to diagnose the prediction of subarachnoid hemorrhage, which can lead to early detection and early treatment of the disease.
- the polymorphism (wild type: T, mutant: C) of the 17th DNA of the 20th intron site DNA sequence of the human elastin gene, and the 23rd intron site DNA sequence A gene having the 24th DNA polymorphism (wild type: ⁇ , mutant type: C) is provided.
- the 17th DNA in the 20th intron site DN ⁇ sequence of the human elastin gene is wild-type (T)
- the 24th DNA in the 23rd intron site DNA sequence is mutated from T to C. Haplotype provided It is.
- a method for determining the presence of a risk of cerebral aneurysm by detecting the eight prototypes in DNA obtained from a specimen. Specifically, the polymorphism of the 17th DNA in the 20th intron DNA sequence of the human elastin gene and the 24th DNA polymorphism of the 23rd intron DNA sequence are identified. The identification provides a method of determining the presence of a risk of developing a cerebral aneurysm.
- a direct nucleotide sequencing method an allele-specific oligonucleotide (AS0) —dot plot analysis, a monobasic primer extension method, a PCR—single-stranded higher-order structure Detecting the presence of the eight prototypes by using any one of the following methods: type (SSCP) analysis, PCR—restriction fragment length polymorphism (RFLP) analysis, invader method and quantitative real-time PCR detection
- SSCP type
- PCR restriction fragment length polymorphism
- invader method quantitative real-time PCR detection
- a method for determining the presence of a risk of cerebral aneurysm which comprises using a single-base primer extension method, specifically, a snapshot (SNaPshot) method or a pyrosequence method. Is done.
- oligo DNA comprising at least a partial sequence of the human elastin gene, wherein the DNA sequence of the 20th intron site DNA sequence is a wild type and the 23rd intron site DNA sequence is a mutant type. can do.
- a gene therapy agent comprising an oligo DNA for converting the 24th nucleic acid sequence of the 23rd intron of the human elastin gene to a wild type as an active ingredient, and a gene using the gene therapy agent Treatment can be provided.
- the present invention there can be provided a method for screening a candidate compound that affects the haplotype of the elastin gene.
- the present invention provides the following inventions.
- Item 1 Identify polymorphisms in the intron part of the human elastin gene Method to determine the presence of a risk of developing a cerebral aneurysm.
- Item 2 The method according to Item 1, which determines the risk of developing a cerebral aneurysm by identifying genetic polymorphisms at the 20th intron and the 23rd intron in the human elastin gene. .
- the 17th DNA in the DNA sequence at the 20th intron of the human elastin gene is wild-type (T)
- the 24th DNA in the DNA sequence at the 23rd intron is the wild-type (T) Item 3.
- Gene polymorphisms can be analyzed by direct nucleotide sequencing, allele-specific oligonucleotide (AS0) -dot blot analysis, single-base primer extension, PCR-single-chain conformational polymorphism (SSCP) analysis, PCR—Identified by any one of items 1 to 3 that is identified using at least one method selected from the group consisting of restriction fragment length polymorphism (RFLP) analysis, invader method, and quantitative real-time PCR detection method Method.
- AS0 allele-specific oligonucleotide
- SSCP PCR-single-chain conformational polymorphism
- Item 5 The method according to Item 4, which is identified using a base primer extension method.
- a primer or probe comprising a combination of the oligonucleotides described in (a) and (b) below.
- an oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is a DNA sequence of the 20th intron site DNA sequence of the human elastin gene;
- an oligonucleotide as a primer or a probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is a nucleotide of the 23rd intron DNA sequence of the 23rd intron of the human elastin gene; Has a sequence containing the polymorphic site of the third DNA, or whether the 3 ′ end of the oligonucleotide is one base more than the polymorphic site of the gene An oligonucleotide that is located a few bases upstream.
- Item 7 Intron region at position 20 of human elastin gene Primer for detecting a polymorphism at DNA position 17 of DNA sequence and intron region 23 of DNA sequence of human elastin gene at position 23 A diagnostic kit for detecting the risk of developing cerebral aneurysms, comprising a primer for detecting a gene polymorphism.
- the 17th DNA in the 20th intron DNA sequence is wild type (T)
- the 24th DNA in the 23rd intron DNA sequence is wild type (T).
- a haplotype comprising a DNA sequence for detecting the risk of developing a cerebral aneurysm and a complementary strand thereof, which is a mutant from () to (C).
- I UPAC-I UB IUPAC- IUB communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984) )
- Guidelines for Preparation of Descriptions Containing Base Sequences or Amino Acid Sequences (edited by the Patent Office) and conventional symbols in the relevant field.
- the term "gene polymorphism” refers to the presence of two or more bases in a certain site in a gene depending on the human.Here, the type frequently used in the general population is different from the wild type. Those having infrequent bases are called mutants.
- a haplotype refers to a combination of gene mutations present in one allele (haploid).
- the eight prototypes of the present invention include the 17th intron site DNA sequence of the 20th intron site of the human elastin gene, named intron 20 / intron 238 protypes, which will be described in Examples described later. Is a wild-type, and a haplotype consisting of a mutant in which the 24th DNA of the 23rd intron site DNA sequence is mutated from T to C. As shown in Figure 4, the human 'elastin gene was reported by Indik et al. To be a 55 kb-long gene consisting of 34 exons. (Bashir, M., et al., J. Biol.
- the present inventor found that the elastin gene was present adjacent to the genetic marker in the 7q1.1.23 region where the strongest linkage was recognized, and conducted direct sequence analysis for gene mutation screening for the gene. (See Example 3). As a result, single nucleotide polymorphisms were detected at 13 sites. Furthermore, the present inventor conducted a case control study to compare allele frequencies for all polymorphisms in order to examine the association between the elastin gene polymorphism and cerebral aneurysms.
- haplotype analysis of the combination of the two polymorphisms revealed that the 17th DNA of intron 20 of the human elastin gene had wild-type T (thymine
- the haplotype in which the 24th DNA of intron 23 was mutated to C (cytosine) showed the strongest significant difference, and found that the combination of both mutations was involved in cerebral aneurysm disease.
- the gene includes not only a double-stranded DNA, but also a single-stranded DNA such as a sense strand and an antisense strand constituting the double-stranded DNA. It is not limited by its length. Therefore, unless otherwise specified, the gene (DNA) of the present invention includes double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), and single-stranded DNA having a sequence complementary to the sense strand. DNA and any of their fragments are included. In the present invention, the gene (DNA) includes a regulatory region, a coding region, an exon, and an intron. In addition, polynucleotides include both RNA and DNA, and genomic DNA NA and synthetic DNA are also included.
- the provision of the eight prototypes 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.
- the eight prototypes of the gene of the present invention can be suitably used, for example, in the development of a novel drug that changes to a wild type.
- the detection of the eight prototypes of the gene of the present invention in an individual or a tissue can be suitably used in elucidation and diagnosis of the cerebral aneurysm.
- the gene or DNA of the present invention can be synthesized by chemical synthesis using the phosphoramidite method or the triester method, or can be performed using a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments are chemically synthesized by synthesizing a complementary strand and either annealing the strands together under appropriate conditions or adding a complementary strand using an appropriate primer sequence and DNA polymerase. It can also be obtained from single stranded products. Eight prototypes of the present invention
- the 17th DNA of the 20th intron site DNA sequence shown in SEQ ID NO: 1 of the human elastin gene is T
- the sequence A haplotype consisting of a DNA sequence in which the 24th DNA of the 23rd intron site DNA sequence shown in No. 2 is mutated from T to C is exemplified.
- the 17th DNA of the 20th intron site DNA sequence of the human elastin gene is T
- the 24th DNA of the 23rd intron site DNA sequence also includes a DNA sequence having a haplotype complementary strand to a sequence comprising a DNA sequence mutated from T to C.
- 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 easily produced and obtained by general genetic engineering techniques based on the sequence information of specific examples of the gene of the present invention disclosed by the present invention.
- a genomic DNA library having eight prototypes of the gene of the present invention is prepared from an appropriate source according to a conventional method, This can be performed by selecting a desired clone using an appropriate probe containing a mutation specific to the gene.
- 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 blood, saliva, lymph, airway mucus, and bodily fluids such as urine and semen. Blood includes serum, plasma and the like.
- the method for screening the gene of the present invention from a genomic DNA library is not particularly limited, and can be performed by a usual method. For example, plaque hybridization, colony hybridization, or a combination thereof using a probe that selectively binds to a target DNA sequence can be exemplified.
- the gene amplification method examples include the PCR method (Saiki, RK, Bugawan, TL, et al., Nature, 324, 163-166 (1986)) and the NASBA method (Co Immediate Tom, J., Nature, 650, 9). Uto 92 (1991)), TMA method (Kacian, DL, and Fultz, TJ, U.S. Patent No. 5,399,491 (1995)) and SDA method (Walker, GT, Little, MC, et al, Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992)).
- a DNA amplification method based on the PCR method [Science, 230, 1350 (1985) 3] can be suitably used (Saiki, RK, Bugawan, TL, et al., Nature, 324, 163-166 (1986)).
- the primer 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 according to the present invention, and can be synthesized according to a conventional method. Further, the isolation and purification of the amplified DNA fragment is performed as described above. Conventional methods can be followed, for example, gel electrophoresis, column purification, and the like. Alternatively, it can be observed with a mass spectrum.
- the eight genes of the gene of the present invention obtained above can be obtained, for example, by using a single-base primer extension method, and the DNA fragment thereof can be obtained by a conventional method, for example, a dideoxy method CProc. Nail. Acad. Sc USA., 74, 5463 ( 1977) 3 or the Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)], or a commercially available sequence kit can be used to determine the nucleotide sequence.
- the gene (haplotype) of the present invention obtained in this way, for example, by utilizing a part or the entire nucleotide sequence of the gene, the presence or absence of the gene of the present invention in an individual or various tissues can be specifically determined. Can be detected. Determination method of the present invention
- the present invention relates to a method for determining the presence of a risk of developing a cerebral aneurysm by identifying a genetic polymorphism of the human elastin gene.
- the method of the present invention is to determine by identifying a genetic polymorphism in the intron portion of the gene.
- the 17th polymorphism at the 20th intron site hereinafter sometimes referred to as “intron 20” and the 23rd intron site (hereinafter referred to as “intron 23”) Identify the 24th polymorphism in) to determine the risk of developing a cerebral aneurysm.
- the method of determining the risk of developing a cerebral aneurysm by identifying the two polymorphisms is not particularly limited as long as the genotype of the gene to be detected is identified. However, for example, the following method can be used.
- a method for obtaining a nucleic acid (DNA) containing a gene encoding human elastin (human elastin gene) from a specimen The DNA may be full-length DNA of human DNA, but is not particularly limited as long as it is at least a partial DNA containing the polymorphic position of the gene of the present invention.
- the DNA is not particularly limited as long as it is derived from humans, and DNA collected from a biological sample containing human DNA, such as blood or a biological material tissue, can be used. DNA may be extracted from these samples by a conventional method, for example, using a commercially available kit or device (QIAGEN Blood & Cell Culture DNA Kit (manufactured by QIAGEN)).
- 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 of the specific sites where the above-mentioned mutation is assumed to be present. It usually has a length of about 50 to several thousand bases, preferably 50 to several hundred bases. In particular, those containing all of the polymorphism sites are preferred.
- DNA obtained by chemically synthesizing a portion containing a mutation based on information on the DNA base sequence of the gene haplotype of the present invention can be generally used.
- a DNA fragment containing the eight prototypes of the gene of the present invention and its mutant bases, which have already been obtained, can also be used favorably.
- a primer set based on the base sequence information of the eight prototypes of the gene of the present invention can be used as a screening probe.
- there is no particular limitation as long as it can hybridize to the sense strand or antisense strand of the human elastin gene without containing the gene polymorphism site and can be used for the following detection methods.
- the nucleotide sequence used as the probe or primer detects 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.
- the positive clone itself having the above sequence can be used as a probe.
- detection of the gene according to the present invention is commonly used for determining the nucleotide sequence of this type of gene, for example, by the dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci. USA, 74). , 5463-5467 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499 (1980)).
- 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.
- a DNA amplification method such as the PCR method described above.
- a gene fragment (sample) amplified by the PCR method is cloned into a plasmid, and then the base sequence is directly sequenced according to the dideoxy method, the Maxa-Mougill-But method, etc. It can be performed by determining the nucleotide sequence using a sequence kit or the like.
- 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.
- an allele-specific oligonucleotide (AS0) dot plot method (Conner, BJ, et al., Proc. Natl. Acad. Sci., 80, 278-282 ( 1983)).
- the method includes, for example, an allele-specific oligonucleotide probe for a gene fragment PCR-amplified using a forward primer designed to sandwich a target single nucleotide polymorphism site and a reverse primer.
- the DNA fragments that hybridize to DNA can be analyzed by dot plot analysis. Thus, it is possible to determine whether a single nucleotide polymorphism is present in the DNA fragment.
- the elastin gene polymorphism (prototype) of the present invention can be detected by a snapshot method (Kuppuswamy, MN, et al. Proc. Natl. Acad. Sci. USA 88, 1143-1147 (1991)), a pyrosequencing method ( Ronaghi, ⁇ ⁇ , et al., Science, 281, 363-365 (1998)), and a single-base primer extension method such as the point mutation detection method disclosed in JP-A-2000-279197. .
- a primer set so as to correspond to the base immediately before or several bases before the target polymorphic site, that is, the 3 'end is set one base upstream or close to the target mutation.
- the primers can also be annealed to the DNA sample.
- the snapshot method can be carried out using ABI PRIMSMNaPshot dNTPPrimerEntenSionKit (manufactured by ABI Biosystems).
- ABI PRIMSMNaPshot dNTPPrimerEntenSionKit manufactured by ABI Biosystems.
- the fluorescent fragments generated after the reaction are analyzed using AB IPRI SM310 / 337/3100/3700 DNA Analyzer (both manufactured by ABI Biosystems) and GeneScan software. Detection and analysis are also possible.
- the pyrosequencing method can be performed, for example, as follows.
- genomic DNA is isolated from a blood sample or the like by a conventional method, PCR-amplified tens to hundreds of bases including the polymorphic site using a biotin-labeled primer, and used to separate the DNA using magnet beads.
- the strand DNA is purified, and the purified DNA is used as a sample.
- a primer set to sequence from a few bases upstream of the desired mutation is allowed to ring with the sample, and then dNTPs are added to the device one by one according to the sequence near the mutation input to the software.
- PPi pyrophosphate
- the PPi is returned to ATP by sulfurylase, and this is used as a substrate for luciferase. Detect using a camera. Gene typing can be performed by analyzing the peak of the destination obtained according to the added ci NTP. Using this method, 96 samples can be typed in about 15 minutes.
- reagents and equipment used in the above method conventional reagents such as DNA polymerase, ATP-sulfurylase, a mixture of four kinds of enzymes of luciferase and apyrase, luciferin and APS (adenosine 5 'sulfate) Phosphoric acid), a commercially available SNP Reagent K its (manufactured by Pyrosequencing AB) comprising dATP (doxyadenosine ⁇ -thio-3phosphate), dCTP, dGTP and dNTP consisting of dTTP ), A PSQ 96 system for automated DNA sequence analysis (manufactured by Pyrosequencing AB), and SNP software for use thereof (PyroSequencing AB) can be used.
- SNP Reagent K its (manufactured by Pyrosequencing AB) comprising dATP (doxyadenosine ⁇ -thio-3phosphate), dCTP, d
- the above pyrosequencing method involves isolating and amplifying a nucleic acid and purifying the amplified PCR product according to the description in, for example, US Pat. No. 6,159,693, and reading the amplified PCR product.
- the reaction can also be carried out by reacting this with pyrophosphoric acid and analyzing the resulting residue. For this overnight analysis, for example, an Exce1 analysis using commercially available READ IT technology (promega Co., Ltd.) can be employed.
- the PCR amplification product (single-stranded DNA) described above is subjected to non-denaturing polyacrylamide gel electrophoresis, and PCR is used to discriminate the presence or absence of a single nucleotide mutation based on the difference in mobility.
- SSCP method Orita, M., Iwahara, II., Et al., Proc. Natl. Acad. Sci. USA, 86, 2776-2770 (1989), Orita, et al., Genomics, 5, 874-879 (1989)).
- restriction enzyme fragment length polymorphism analysis is performed.
- the method (RFLP method: Botstein, DR, et al., Am. J. Hum. Gen., 32, 314-331 (1980)) may be used.
- the RFLP method is a PCR-RFLP method, that is, after amplifying and preparing a sample DNA by a PCR method or a modified method thereof in advance, the RFLP method is performed on a large amount of a prepared and concentrated sample DNA.
- the method of implementation is used. Thus, the presence or absence of a specific cleavage site can be detected.
- the detection of the eight prototypes 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, the DNA of the elastin gene is extracted from a human biological sample, the DNA fragment of the region containing the gene polymorphism site of intron 20 and / or 23 of the gene is amplified, and a large and concentrated sample sample is obtained. obtain. Next, the amplified DNA sample is digested with a specific restriction enzyme (ie, an enzyme capable of digesting either the wild type or the mutant type only), and the DNA cleavage mode (such as presence or absence of cleavage, base length of the cleavage fragment, etc.) ) Is checked in accordance with the usual method.
- a specific restriction enzyme ie, an enzyme capable of digesting either the wild type or the mutant type only
- the DNA cleavage mode such as presence or absence of cleavage, base length of the cleavage fragment, etc.
- Detection of the genetic polymorphism or haplotype of the elastin gene of the present invention can also be carried out by the following Invader method and the quantitative real-time PCR detection method (TaqMan method).
- the method is a method in which it is not necessary to amplify a target DN in advance to analyze a single nucleotide polymorphism in a genomic DN, and is carried out as follows.
- the first consists of a 5 'flap of 5 to 50 bases in length and an oligonucleotide of 30 to several hundred bases synthesized to complement the target genomic DNA by placing the nucleic acid to be detected at the 3' end of the 5 'flap.
- Integrator consisting of 15 to several tens of nucleotides synthesized to complement target genomic DNA, except that the target probe and the nucleic acid complementary to the nucleic acid to be detected are placed at the 3 'end.
- Oligonucleotides ⁇ Probes are synthesized by, for example, 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 cleaves the 5 ′ flap of the first probe is added.
- the genomic DNA in the sample contains the desired nucleic acid for detection
- the 5 ′ flap having the nucleic acid for detection at the 3 ′ end is released by cleavage with the restriction enzyme, The first reaction ends. 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 continues by acting as an Invader' oligo in a second reaction using a fluorescence resonance energy transfer (FRET) probe as the target.
- FRET fluorescence resonance energy transfer
- the 5 'flap on the first probe except for the base at the 3' end, is complementary to the FRET probe used in the second reaction.
- Each FRET probe used in the second reaction is then constructed to contain the same sequence and consist essentially of two elements, regardless of the target to be detected. (1) a 3 'region complementary to the product cleaved from the first reaction, and (2) a duplex to mimic the stranded probe, and the targets hybridize together and become It has a self-complementary region containing an overnight fluorescent dye and a quencher fluorescent dye.
- the fluorescence intensity of the reporter fluorescent dye is suppressed by fluorescence resonance energy transfer, and the quencher fluorescent dye is If the probe is not bound to the same probe, the fluorescence intensity is not suppressed. Therefore,
- restriction enzyme cleavage of the FRET probe separates the two fluorescent dyes and produces a detectable fluorescent signal.
- the combination of the first and second reactions in which the standard fluorescence microtiter plate reader can read and detect the product, can amplify the signal from 1 to 1 ⁇ 10 6 fold.
- the presence or absence of a desired wild-type or mutant-type nucleic acid or a haplotype can also be detected by using the Invader-Attsey method.
- the detection of the genetic polymorphism of the elastin gene of the present invention can also be easily performed by a quantitative real-time PCR detection method (TaqMan method).
- the method can be performed 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 bases to about 30 bases are prepared. However, both the forward primer and the reverse primer should be prepared so as not to include a nucleic acid site for detecting the presence or absence of a target mutation.
- a region in which a repo overnight fluorescent dye and a quencher fluorescent dye are bound to an oligonucleotide having a base sequence consisting of 15 to 50 bases, and where the forward primer hybridizes Create a probe that selects a combination in which the region that the probe hybridizes with 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 nucleotide nucleic acid mutation.
- PCR is performed using the desired DNA fragment region of the elastin gene to be measured in the sample as type III, and the fluorescence detected from the reaction solution is measured in real time.
- the presence or absence of the mutation can be detected.
- a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable.
- FAM 6-carboxy-fluorescein
- Mono-damine fluorescent dyes such as R A (6-potoxy-tetramethyl-rhodamine) are preferred. These fluorescent dyes are known and can be used because they are included in a commercially available kit for real-time detection PCR.
- the binding positions of the repo overnight fluorescent dye and the quencher fluorescent dye are not particularly limited, but usually, a reporter fluorescent dye is attached to one end (preferably 5, terminal) of the oligonucleotide portion of the probe, and the other end is attached to the other end. Quencher-fluorescent dye is bound.
- Methods for binding a fluorescent dye to an oligonucleotide are known, for example, Noble et al., Nuc. Acids Res. 12: 3387-3403 (1984) and Iyer et al., J. Am. Chem.
- the real-time detection PCR method itself is known, and devices and kits for the method are also commercially available, such commercially available devices and kits can be used in the present invention.
- the method can be carried out according to the method described in Japanese Patent No. 2825976, or according to the AB PRISM 7700 Sequencing System User Manual manufactured by PE Biosystems.
- Detection of SNPs or haplotypes of the elastin gene of the present invention can also be carried out by the following various methods generally known as DNA base sequence determination methods or mutation detection methods.
- PCR-SSO method using sequence-specific oligonucleotides A probe for each mutation was immobilized on a carrier, and a sample (gene amplification product) was hybridized to the carrier, and hybridization was determined based on the presence or absence of mismatch. A method to determine the difference in efficiency.
- PCR—DGGE (denaturing gradient gel electrophoresis) method After hybridizing the fragment and the normal DNA fragment and performing electrophoresis on a polyacrylamide gel in which the concentration of denaturing agents such as urea and formamide is gradually increasing, a homoduplex without mismatches is obtained. In comparison, dissociation into single strands occurs at lower concentrations of denaturants. Since single-stranded DNA has a higher electrophoresis speed than double-stranded DNA, a single-base mutation can be detected by comparing the difference in mobility.
- DGGE denaturing gradient gel electrophoresis
- a method that utilizes the property of recognizing and cleaving hetero-double-stranded DNA containing single base mismatches such as deletion and insertion of T4 endonuclease, and which contains DNA in a test sample containing a mutation After PCR-amplifying the mixture of the fragment and the labeled sequence-specific probe, the DNA fragment is cleaved at the mutation site with T4 endonuclease, and further reacted with another sequence-specific probe and the cleaved DNA.
- the DNA fragments are again cut at the mutation site by T4 endonuclease, and the cleavage products separated by each cleavage are electrophoresed in polyacrylamide gel, and the generated electrophoretic patterns are compared to obtain a target. It can detect whether two mutations are present in the test sample.
- the haplotype of the polymorphic gene be determined, but also the paternal haplotype and the maternal haplotype can be specified from the DNA sample.
- preferred methods include a single-base primer extension method, specifically, a snapshot method or a pyrosequencing method.
- the primer used in the gene detection method is not particularly limited as long as it can specifically amplify only the region containing the haplotype of the gene of the present invention, and is appropriately designed based on the sequence information of the gene of the present invention. be able to.
- those having a length of about 15 to 50 nucleotides and having a nucleotide sequence synthesized so as to sandwich the SNP of each of the eight prototypic sequences of the gene of the present invention are exemplified.
- the above-mentioned primers having a 3 ′ end one to several bases upstream (5 ′ side) in the sense strand or the antisense strand from the single nucleotide polymorphism site may be used.
- the above detection method may be appropriately set so that the gene polymorphism site of intron 20 and the gene polymorphism site of intron 23 can be simultaneously identified. In addition, it may be set so that each gene polymorphism can be identified separately.
- the gene haplotype of the present invention includes a specific primer and a DNA used as a specific probe for detecting the polymorphic gene according to the present invention. Fragments are also included.
- elastin is used as a gene-specific probe for detection.
- 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.
- the present invention provides a primer for detecting a polymorphism in the 17th DNA of the DNA sequence of the 20th intron of the human elastin gene, and a 23rd intron of the human elastin gene. It is intended to provide a diagnostic kit for detecting the risk of developing a cerebral aneurysm, comprising a primer for detecting a gene polymorphism of the 24th DNA of the site DNA sequence.
- each primer capable of hybridizing to the sense strand or antisense strand of the elastin gene and capable of amplifying each of the gene polymorphism sites of intron 20 and intron 23, a gene polymorphism site
- examples of such primers include a primer having a sequence having a 3 ′ end one to several bases upstream (5 ′ side) from the base and a primer having a sequence having a gene polymorphism site.
- the present invention provides a reagent kit for detecting the risk of developing a human cerebral aneurysm.
- the reagent kit contains, as an essential component, a DNA fragment that at least partially hybridizes to a part or all of the DNA base sequence of at least intron 20 and intron 23 of the elastin gene or its complementary base sequence. If present, labeling agents and other reagents essential for PCR (eg, Taq
- DNA polymerase deoxynucleotide triphosphates, primers, etc.
- the labeling agent examples include a chemical modifying substance such as a radioisotope or a fluorescent substance, and the DNA fragment itself may be conjugated with the labeling agent in advance.
- the reagent kit may include a reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., which are appropriate for the benefit of performing the measurement.
- the present invention also provides a method for diagnosing the risk of developing human cerebral aneurysm using the above-mentioned measurement method, a diagnostic agent used for the method, and a diagnostic kit.
- Gene therapy of the present invention is a method for diagnosing the risk of developing human cerebral aneurysm using the above-mentioned measurement method, a diagnostic agent used for the method, and a diagnostic kit.
- the present invention provides an oligo DNA for converting the 24th nucleic acid sequence of intron 23 of the human elastin gene into a wild type, or a gene therapy vector containing the oligo DNA and a human
- An object of the present invention is to provide a drug containing, as an active ingredient, cells into which oligo DNA for converting the nucleic acid sequence at position 24 of intron 23 of the elastin gene to wild type has been introduced.
- an oligo comprising several tens to several hundreds of bases, comprising a partial sequence of the human elastin gene, and being synthesized so that the 24th nucleic acid sequence of intron 23 is T Nucleotides, transfer vectors for gene therapy containing the oligonucleotides, cells transfected with the oligonucleotides with the vectors, and introns of the human elastin gene with the transfer vectors for gene therapy or the vector.
- a gene therapy agent comprising a cell into which the wild-type nucleic acid is introduced as an active ingredient.
- a gene therapy drug containing a cell into which a wild-type nucleic acid has been introduced as an active ingredient is a mutation in which the 17th nucleic acid of intron 20 of the elastin gene is wild-type and the 24th nucleic acid of intron 23 is mutated to C.
- a cerebral aneurysm having a haplotype or a patient with a cerebral aneurysm (subarachnoid hemorrhage) having the haplotype, or to a peripheral blood tissue site of the patient.
- the practice of the present invention can employ, unless otherwise indicated, conventional methods of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology.
- Mania is, T., et al., Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, cold Spring Harbor, New York (1982)), Sambrook, J., et al., Molecular Cloning a Laboratory Manual, 2nd Ed.
- Gene analysis for detecting the eight elastin gene prototypes of the present invention can be performed by the above-described determination method and the like.
- genomic DNA obtained from cells from human blood or tissue cells was modified to include the elastin gene intron 20 17 and intron 23 24 nucleic acid sequences, respectively.
- the presence of the eight human elastin gene prototype can be confirmed.
- Suitable methods include a method of extracting blood and extracting DNA from blood cells, and a method of extracting DNA from cells obtained directly from target tissue cells.
- the following gene therapy is performed.
- Suitable vectors here can be made using the vectors disclosed in US Pat. Nos. 5,252,479 and PCT International Publication WO 93/07282.
- the prepared vector containing DNA in which the intron 23 gene region of the human elastin gene contains wild-type DNA is injected into cells such as blood, and administered to the target patient systemically. If the transformed gene is not permanently integrated into the chromosome of the targeted vascular cell, the process can be repeated periodically.
- 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, the gene is introduced outside the body, and then the cell is introduced into the body Any of the ex vivo methods of returning can be appropriately selected and used.
- the DNA in which the intron 23 of the elastin gene to be introduced is wild-type can be obtained by general genetic engineering based on the base sequence information of the gene. Easily manufacture and acquire by method Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Seismic Chemistry Laboratory Lecture “Gene Research Methods I, II, III”, edited by The Biochemical Society of Japan (1986), etc.]. The method described above can be applied to the synthesis of DNA.
- a biological sample such as blood or serum is prepared, nucleic acids are extracted as desired, and whether or not the octaprototype-sensitive genes of human elastin gene intron 20 and intron 23 are present. It is also possible to analyze
- the presence of a risk of cerebral aneurysm development can be detected by analyzing the haplotypes of intron 20 and intron 23 of the elastin gene obtained from a sample such as blood or serum.
- FIG. 1 is a drawing showing the maximum power and maximum 1 od by multipoint linkage analysis on chromosome 5.
- FIG. 2 is a drawing showing one marker and a maximum of 1 od by a multipoint linkage analysis on chromosome 7.
- FIG. 3 is a drawing showing a marker of chromosome 14 by multipoint linkage analysis and a maximum of 1 od.
- FIG. 4 is a drawing showing the structure of the elastin gene (ELN).
- EPN elastin gene
- PM stands for motor
- EX stands for exon
- INT stands for intron.
- FIG. 5 is a drawing showing the 17th DNA polymorphism (wild type: T, mutant type: C) in the DNA sequence at the 20th intron site of the human elastin gene.
- FIG. 6 is a drawing showing the polymorphism of the 24th DNA in the DNA sequence of the 23rd intron of the human elastin gene (wild type: ⁇ , mutant type: C).
- the principle of the method is as follows. Since the affected sibling inherits the allele that causes the disease from the parent, the allele must be shared, while the number of alleles shared by the sibling 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 has been recognized".
- Linkage mapping set version I I. PE Applied Biosystems, Inc. consisting of 400 fluorescently labeled microsatellites for the purpose of linkage analysis covering the entire genome was used. According to this marker set, analysis can be performed at an average interval of 8.6 centiMorgan. However, since there was a marker with low heterozygosity in Japanese, the number of markers was further increased, and analysis of the entire genome was performed using 420 markers.
- a highly dimorphic dinucleotide repeat (dinuc) Leotide repeat microsatellite) could be amplified by PCR.
- the difference in the size of the PCR product thus obtained was confirmed on a sequence gel using an automatic analyzer ABI PRISM: Model 377 (manufactured by Applied Biosystems). Since the PCR reaction is performed using the primers labeled with the above three kinds of fluorescent dyes, even if the PCR products have the same size, the three kinds of colors can be distinguished separately. With the above-mentioned reaction gel, 96 kinds of samples could be analyzed simultaneously for 15 kinds of markers, and about 1500 genotyping could be processed by one electrophoresis.
- the linkage test is performed using the nonparametric analysis GENEHUNTER (KruglyaK, L. ei al. Am. J. Genet., 58, 1347-1363 (1996)), and the SIBPAL program (SIBPAL: SAGE, Statistical Analysis for Genetic Epidemiology, release 3.1. Department of Biometry and Genetics. LSU Medical Center, New Jersey, LA). Analysis result:
- chromosome 5 had a maximum lod of 2.24
- chromosome 7 had a maximum of 1 od 3.22 as shown in Figure 2
- chromosome 14 had a maximum of od 3.22 as shown in Figure 3. 1 od 2.31 was obtained.
- P 0.000027 for the marker D7S2472 on chromosome 7.
- Elastin is at the core of the elastin fiber, 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 Cell Biology (Tokyo Kagaku Dojinsha, 1997)). The most common site of cerebral aneurysms 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);). As a result of linkage analysis, and from a known technique regarding the functional role of elastin, the elastin gene was considered to be a potential disease candidate gene for cerebral aneurysms.
- the elastin gene is located at 7Q11.23 as described above, A gene consisting of 34 exons with a total length of 55 kilobases located at 86.5 centiMorgans (cM) from the parent (Peoples, R., et al. Am. J. Hum. Genet. 66 (1) 7-68 (2000)).
- the target patients had a cerebral aneurysm with a diameter of 4 or more by angiography, MRA (magnetic resonance angiography), or 3 DCTA (3 dimentional computed tolographic angiography).
- Non-patients control were those without cerebral aneurysm according to the method described above.
- Chi-tests X-tests were performed for each polymorphism in 168 patients and 188 non-patients, and allele frequencies were compared between patients and controls.
- those having these intron 20 and intron 23 polymorphisms have a high probability of being a human or patient having a risk of developing a cerebral aneurysm.
- a human having a haplotype in which the 17th intron 20 of elastin gene is intron 20 and the 24th T of intron 23 is mutated to C has a high risk of developing a cerebral aneurysm.
- the relative risk (odds rat io) is 1.91, ie, humans with this haplotype are 1.9 times more cerebral aneurysms than those without this haplotype Susceptible to the disease.
- intron 20 (17th T ⁇ C) and intron 23 (24th T ⁇ C) were measured. 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.
- a snapshot in which a single base extension reaction is performed is preferably exemplified.
- This method can be simply measured by using ABPIPRISMSNaPshotddNTPPrimerExtensionKit (manufactured by ABI Nano Systems). Fluorescence fragments generated after the reaction can be analyzed with ABI PRIMSM310NO377Z3100 / 3700 and GeneScan software as well.
- the nucleotide to be hybridized to the antisense strand is a wild-type of any length, for example, about 16 to 24 nucleotides in length from the DNA sequence No. 16 at the intron 20 site to the 5 'side of the sense strand. Create a primer consisting of the type sequence.
- a primer consisting of a wild-type sequence of an arbitrary length, for example, about 16 to 24 bases in length from the DNA sequence number 18 at the intron 20 to the 5 'side of the antisense strand, such as intron 20, is used.
- DNA sequence (SEQ ID NO: 1) Primer consisting of a nucleic acid sequence of 20 bases complementary to the sequence of Nos. 18 to 37 (SEQ ID NO: 1) 3) is synthesized. Then, mix 6-FAM and ddNTP labeled with different colors of fluorescence of ATGC such as HEX.
- d dNTP is a base that stops elongating by one base extension
- the primer that has extended only one base will have a color depending on the type of base at the extended end as a result of this reaction. That is, it can be determined whether the 17th DNA sequence of the 20 introns is a wild type or a mutant type. Similarly, it is possible to determine whether the 24th DNA of intron 23 is wild type or mutant type.
- a primer SEQ ID NO: 4 consisting of a 20-base long nucleic acid sequence complementary to the sequence of ID Nos. 25 to 44 in the DNA sequence of Intron 23 (SEQ ID NO: 2) is synthesized and discriminated in the same manner as described above.
- the primers fluorescently labeled with this ddNTP are separated by electrophoresis, and the fluorescent signal is analyzed and detected.
- a method of detecting a fluorescent signal by electrophoresis a method of detecting a difference in molecular weight accompanying extension of one base with a mass spectrum can also be applied.
- Example 5-2 Method for Detecting Risk of Onset of Cerebral Aneurysm
- PP i Pyrophosphate
- CCD camera a substrate for luciferase. Since a luminescence peak is obtained according to the added dNTP, that is, the nucleotide sequence, genotyping becomes possible. With this method, 96 samples could be typed in about 15 minutes.
- the above method is a substrate solution consisting of DNA enzyme, ATP-sulfurylase, luciferase, four kinds of enzyme mixture of apyrase, luciferin, APS (adenosine 5 'sulfate phosphate), d ATP ( Using commercially available SNPR eag ent K its (manufactured by Pyrosequencing AB) having dNTP consisting of doxyadenosine ⁇ -thio-3-phosphate), dCTP, dGTP and dTTP as components, The measurement can be carried out simply using a P SQ 96 system (manufactured by Pyrosequencing AB) for automatic DNA sequence analysis and SNP software (manufactured by Pyrosequencing AB) for use.
- P SQ 96 system manufactured by Pyrosequencing AB
- SNP software manufactured by Pyrosequencing AB
- the human elastin gene intron 20 and intron 23 WV eight prototypes found by the present invention has a high risk of developing a cerebral aneurysm, It is possible to easily detect the presence of human cerebral aneurysm development risk.
- the method for detecting a risk of developing a cerebral aneurysm according to the present invention, it is possible to establish a secondary prevention system in which a person having a genetic risk of a cerebral aneurysm is identified and diagnosed before bleeding. . Further, by using the present invention, it is also possible to appropriately select a target for a brain dock or an operation.
- a gene haplotype associated with a risk of developing a human cerebral aneurysm there is provided a gene haplotype associated with a risk of developing a human cerebral aneurysm.
- the gene eight prototype of the present invention has a linkage in human cerebral aneurysm development. It is strongly considered to be a factor that promotes the development of cerebral artery blood vessels in these cells and their surrounding tissues, especially subarachnoid hemorrhage. '
- the analysis of the polymorphism at a specific site in intron 20 of the human elastin gene and the polymorphism at a specific site in intron 23 ⁇ is a study on the relationship between gene function and onset of cerebral aneurysm.
- a gene transfer vector useful for gene therapy comprising a wild-type oligonucleotide at the intron 23 site of the human elastin gene, a wild-type at the intron 23 site of the human elastin gene. Also provided are a cell into which an oligonucleotide has been introduced, a gene therapy agent comprising the vector or the cell as an active ingredient, and a gene therapy method utilizing the same.
- a cerebral aneurysm (subarachnoid) caused by a polymorphism at a specific site of intron 20 and a polymorphism at a specific site of intron 23 of the human elastin gene is caused by octaprobe. It is also possible to provide a screening method of a candidate compound that reduces the risk of the occurrence of (bleeding), and a screening kit.
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Abstract
A method of judging the onset risk of brain aneurysm by identifying the gene polymorphism of the intron of human elastin gene.
Description
明 細 書 Specification
脳動脈瘤感受性遺伝子 技 術 分 野 Cerebral aneurysm susceptibility gene Technology field
本発明は、 脳動脈瘤 （クモ膜下出血） に関与する遺伝子、 および該遺伝子 の利用に関する。 The present invention relates to a gene involved in cerebral aneurysm (subarachnoid hemorrhage) and use of the gene.
背 景 技 術 Background technology
「国民衛生の動向」 によると、 脳血管疾患 (脳卒中）の死亡率は、 高血圧症 管理の徹底、 生活環境の改善などにより昭和 4 0年代以降順調に低下し、 昭 和 6 0年には第三位となった。 しかし脳卒中死亡率は低下したものの、 治療 方法の進歩、 軽症例の増加によりその患者総数は、 著しく増加し、 いわゆる 「寝たきり」 の最大の原因となっている。 脳卒中の中でもクモ膜下出血の死 亡率は昭和 2 6年より一貫して漸増している。 手術療法の目覚しい進歩があ るものの手術対象となるのはクモ膜下出血患者の一部で、 約半数の患者は初 回出血で、 死亡するためである。 特に高齢者でのクモ膜下出血は重症例が多 く、 手術成績も不良で、 「寝たきり」 にいたるケースが多い。 そのため、 早 期発見、 早期治療（出血前に発見し治療する）を行なう二次予防が、 是非必要 とされている。 しかしながら、 早期発見、 早期治療のために、 例えば高血圧 症の管理の他に何を指標するかについて、 未だ定かではない現状にある。 According to the “Trends in National Health”, the mortality rate of cerebrovascular disease (stroke) has been steadily decreasing since the 1940s due to the thorough management of hypertension and the improvement of the living environment. Third place. However, although stroke mortality has declined, advances in treatment and the number of mild cases have led to a significant increase in the total number of patients, which is the largest cause of so-called bedridden. The death rate of subarachnoid hemorrhage among strokes has been gradually increasing since 1926. Despite the remarkable progress in surgical therapy, only a few patients with subarachnoid hemorrhage will be treated, and about half of them will die from the first bleeding. In particular, subarachnoid hemorrhage in the elderly is often severe, with poor surgical results, often leading to bedridden. Therefore, early prevention, and secondary prevention of early treatment (detection and treatment before bleeding) is definitely needed. However, for early detection and early treatment, for example, it is still unclear what indicators to index in addition to the management of hypertension.
一方、 これまでの疫学調査により、 脳動脈瘤の発生には、 比較的強い遺伝 背景があることが知られている。 全国 1 1 0 0ケ所の脳神経外科関連施設の 協力により、 1 0 4対の罹患同胞対（脳動脈瘤患者同胞）が集められた。 8 5 家系収集され、 その内訳は、 7 7組の 2人兄弟、 7組の 3人兄弟、 1組の 4 人兄弟である。 また、 7 3家系で発端者がクモ膜下出血であり （脳動脈瘤破 裂確認済み） 、 1 2家系で発端者が未破裂脳動脈瘤のみを認めている（Kasuya, H.， e t al . , Neurosurgery, 46 (6) 1301-1306 (2000) ) . 遺伝要因の強さの指 標である、 兄弟での相対危険率 （A s) は 6とされている。 On the other hand, epidemiological studies so far have shown that the occurrence of cerebral aneurysms has a relatively strong genetic background. With the cooperation of 110 neurosurgery-related facilities nationwide, 104 affected sib pairs (branch aneurysm patient siblings) were collected. 8 5 families were collected, consisting of 77 pairs of 2 brothers, 7 pairs of 3 brothers and 1 pair of 4 brothers. The proband had subarachnoid hemorrhage in 73 families (ruptured cerebral aneurysm was confirmed), and the proband had only unruptured cerebral aneurysm in 12 families (Kasuya, H., et al. , Neurosurgery, 46 (6) 1301-1306 (2000)). The relative risk factor (As) for siblings, which is an indicator of the strength of genetic factors, is considered to be 6.
脳動脈瘤は遺伝的要因と環境的要因が複雑に関与して発症に至ると考えら
れているが、 脳動脈瘤の責任遺伝子は未だ知られていない。 発 明 の 開 示 Cerebral aneurysms may be complicated by genetic and environmental factors However, the genes responsible for cerebral aneurysms are still unknown. Disclosure of the invention
本発明者は、 脳動脈瘤の責任遺伝子を明らかにすべく、 連鎖解析による遺 伝子座の同定を行い、 その結果、 脳動脈瘤の発生との関連性の高い責任遺伝 子を見出した。 The present inventors identified loci by linkage analysis in order to clarify the genes responsible for cerebral aneurysms, and as a result, found a responsible gene highly relevant to the occurrence of cerebral aneurysms.
本発明の課題は、 脳動脈瘤 （クモ膜下出血） と関連性の高い責任遺伝子の 同定にある。 該遺伝子の同定により、 脳動脈瘤の遺伝的リスクを有するヒト を同定し、 脳出血を起こす前に診断する二次予防の体制を確立することが可 能となる。 An object of the present invention is to identify a responsible gene highly associated with cerebral aneurysm (subarachnoid hemorrhage). By identifying the gene, it is possible to identify a person who has a genetic risk of cerebral aneurysm and establish a secondary prevention system for diagnosing cerebral hemorrhage before it occurs.
本発明者は、 上記課題を解決することを主な目的として鋭意研究を重ねた 結果、 脳動脈瘤罹患同胞対連鎖解析により脳動脈瘤遺伝子座のひとつを 7 q 1 1. 23の領域に特定し、 有力な候補遺伝子として、 エラスチン遺伝子を 見出した。 エラスチン遺伝子のェキソン 20 (Nucleic Acids Research 19, 4314, 1991) 及び 3UTR 1 (Human Gent., 104, 135-142,1999) に遺伝子 多型が存在することは既に知られていたが、 本発明者は更に研究を重ね、 ェ ラスチン遺伝子の遺伝子多型と脳動脈瘤との関連性を八プロタイプ解析によ り検討した。 その結果、 該エラスチン遺伝子のイントロン部分、 具体的には イントロン 20とイントロン 23ハプロタイプが、 患者において有意に頻度 が高いことが判明し、 このイントロン 20とイントロン 23の組み合わせの 遺伝子タイピングにより、 脳動脈瘤の早期発見、 早期治療に結びつく、 クモ 膜下出血の罹患予測の診断ができることを見出した。 The present inventors conducted intensive research with the main aim of solving the above problems, and as a result, identified one of the cerebral aneurysm loci in the region of 7q1.1.23 by linkage analysis of siblings with cerebral aneurysm Then, the elastin gene was found as a promising candidate gene. It was already known that the gene polymorphism exists in exon 20 (Nucleic Acids Research 19, 4314, 1991) and 3UTR 1 (Human Gent., 104, 135-142, 1999) of the elastin gene. Conducted further studies and examined the association between the genetic polymorphism of the elastin gene and cerebral aneurysm by eight-prototype analysis. As a result, it was found that the intron portion of the elastin gene, specifically, the intron 20 and intron 23 haplotypes were significantly higher in patients, and the genotyping of the combination of intron 20 and intron 23 showed that It has been found that it is possible to diagnose the prediction of subarachnoid hemorrhage, which can lead to early detection and early treatment of the disease.
即ち、 本発明によれば、 ヒト ·エラスチン遺伝子の 20番目のイントロン 部位 DNA配列の 17番目の DNAの遺伝子多型 （野生型： T、 変異型： C) 、 且つ 23番目のイントロン部位 DNA配列の 24番目の DNAの遺伝 子多型 （野生型： Τ、 変異型： C) を有する遺伝子が提供される。 That is, according to the present invention, the polymorphism (wild type: T, mutant: C) of the 17th DNA of the 20th intron site DNA sequence of the human elastin gene, and the 23rd intron site DNA sequence A gene having the 24th DNA polymorphism (wild type: Τ, mutant type: C) is provided.
また、 ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DN Α配列の 17番目の DNAが野生型 （T) であって、 且つ 23番目のイントロン部位 DNA配列の 24番目の DN Aが Tから Cに変異したハプロタイプが提供さ
れる。 In addition, the 17th DNA in the 20th intron site DNΑ sequence of the human elastin gene is wild-type (T), and the 24th DNA in the 23rd intron site DNA sequence is mutated from T to C. Haplotype provided It is.
本発明によれば、 検体から得られた D N Aにおいて、 前記八プロタイプを 検出することによって脳動脈瘤のリスクの存在を判定する方法が提供される。 具体的には、 ヒト ·エラスチン遺伝子の 2 0番目のイントロン部位 D N A配 列の 1 7番目の D N Aの遺伝子多型及び 2 3番目のイントロン部位 D N A配 列の 2 4番目の D N Aの遺伝子多型を同定することにより、 脳動脈瘤の発症 のリスクの存在を判定する方法が提供される。 According to the present invention, there is provided a method for determining the presence of a risk of cerebral aneurysm by detecting the eight prototypes in DNA obtained from a specimen. Specifically, the polymorphism of the 17th DNA in the 20th intron DNA sequence of the human elastin gene and the 24th DNA polymorphism of the 23rd intron DNA sequence are identified. The identification provides a method of determining the presence of a risk of developing a cerebral aneurysm.
さらに本発明によれば、 該前記方法のために、 ヌクレオチド直接配列決定 法、 対立遺伝子特異的ォリゴヌクレオチド（AS0)—ドットプロット分析、 一塩 基プライマー伸長法、 P C R—単鎖高次構造多型（SSCP)分析、 P C R—制限 酵素断片長多型（RFLP)分析、 ィンベーダ一法および定量的リアルタイム P C R検出法のいずれかの方法を用いることを特徴とする、 前記八プロタイプの 存在を検出することによつて脳動脈瘤のリスクの存在を判定する方法が提供 される。 好ましくは、 一塩基プライマー伸長法、 具体的には、 スナップショ ット（SNaPshot)法またはピロシーケンス（Pyrosequenc ing)法を用いることを 特徴とする脳動脈瘤のリスクの存在を判定する方法が提供される。 Furthermore, according to the present invention, for the method, a direct nucleotide sequencing method, an allele-specific oligonucleotide (AS0) —dot plot analysis, a monobasic primer extension method, a PCR—single-stranded higher-order structure Detecting the presence of the eight prototypes by using any one of the following methods: type (SSCP) analysis, PCR—restriction fragment length polymorphism (RFLP) analysis, invader method and quantitative real-time PCR detection Thus, a method for determining the presence of a risk of cerebral aneurysm is provided. Preferably, there is provided a method for determining the presence of a risk of cerebral aneurysm, which comprises using a single-base primer extension method, specifically, a snapshot (SNaPshot) method or a pyrosequence method. Is done.
また、 本発明によれば、 少なくともヒト ·エラスチン遺伝子の部分配列で あって、 2 0番目のイントロン部位 D N A配列が野生型および 2 3番目のィ ントロン部位 D N A配列が変異型であるオリゴ D N Aを提供することができ る。 Further, according to the present invention, there is provided oligo DNA comprising at least a partial sequence of the human elastin gene, wherein the DNA sequence of the 20th intron site DNA sequence is a wild type and the 23rd intron site DNA sequence is a mutant type. can do.
さらに本発明によれば、 ヒト ·エラスチン遺伝子の 2 3番目のイントロン の 2 4番目の核酸配列を野生型に変換するためのオリゴ D N Aを有効成分と する遺伝子治療剤および該遺伝子治療剤を用いる遺伝子治療法を提供するこ とができる。 Further, according to the present invention, a gene therapy agent comprising an oligo DNA for converting the 24th nucleic acid sequence of the 23rd intron of the human elastin gene to a wild type as an active ingredient, and a gene using the gene therapy agent Treatment can be provided.
また、 本発明によれば前記エラスチン遺伝子のハプロタイプに影響を与え る候補化合物のスクリーニング方法を提供することができる。 本発明は、 以下の発明を提供するものである。 Further, according to the present invention, there can be provided a method for screening a candidate compound that affects the haplotype of the elastin gene. The present invention provides the following inventions.
項 1 . ヒト 'エラスチン遺伝子のイントロンの部分の遺伝子多型を同定す
ることにより、 脳動脈瘤の発症のリスクの存在を判定する方法。 Item 1. Identify polymorphisms in the intron part of the human elastin gene Method to determine the presence of a risk of developing a cerebral aneurysm.
項 2. ヒト ·エラスチン遺伝子における、 20番目のイントロン部位と 2 3番目のイントロン部位の遺伝子多型を同定することにより、 脳動脈瘤の発 症のリスクの存在を判定する項 1に記載の方法。 Item 2. The method according to Item 1, which determines the risk of developing a cerebral aneurysm by identifying genetic polymorphisms at the 20th intron and the 23rd intron in the human elastin gene. .
項 3. ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DN A配列の 17番目の DNAが野生型 （T) であって、 且つ 23番目のイントロン部位 DNA配列の 24番目の DNAが野生型 （T) から （C) に変異した変異型 である遺伝子多型を同定することにより、 脳動脈瘤の発症のリスクの存在を 判定する項 1又は 2のいずれかに記載の方法。 Item 3. The 17th DNA in the DNA sequence at the 20th intron of the human elastin gene is wild-type (T), and the 24th DNA in the DNA sequence at the 23rd intron is the wild-type (T) Item 3. The method according to any one of Items 1 or 2, wherein the presence of a risk of cerebral aneurysm is determined by identifying a gene polymorphism that is a variant mutated from (C) to (C).
項 4. 遺伝子多型を、 ヌクレオチド直接配列決定法、 対立遺伝子特異的ォ リゴヌクレオチド（AS0)—ドットブロット分析、 一塩基プライマー伸長法、 P CR-単鎖高次構造多型（SSCP)分析、 P C R—制限酵素断片長多型 (RFLP)分 析、 ィンベーダ一法および定量的リアルタイム P C R検出法からなる群から 選ばれる少なくとも一つの方法を用いて同定する項 1乃至 3のいずれかに記 載の方法。 Item 4. Gene polymorphisms can be analyzed by direct nucleotide sequencing, allele-specific oligonucleotide (AS0) -dot blot analysis, single-base primer extension, PCR-single-chain conformational polymorphism (SSCP) analysis, PCR—Identified by any one of items 1 to 3 that is identified using at least one method selected from the group consisting of restriction fragment length polymorphism (RFLP) analysis, invader method, and quantitative real-time PCR detection method Method.
項 5. —塩基プライマ一伸長法を用いて同定する項 4に記載の方法。 Item 5. —The method according to Item 4, which is identified using a base primer extension method.
項 6. 以下の （a) 及び （b) に記載のオリゴヌクレオチドの組み合わせ からなるプライマ一又はプローブ。 Item 6. A primer or probe comprising a combination of the oligonucleotides described in (a) and (b) below.
(a) ヒト ·エラスチン遺伝子にハイブリダィズすることができる遺伝子多 型検出用プライマー又はプロ一ブとしてのオリゴヌクレオチドであって、 該 オリゴヌクレオチドが、 ヒト ·エラスチン遺伝子の 20番目のイントロン部 位 DNA配列の 17番目の DN Aの遺伝子多型部位を含む配列を有するか、 又は、 該オリゴヌクレオチドの 3 ' 末端が該遺伝子多型部位よりも 1塩基か ら数塩基上流に位置するものであるオリゴヌクレオチド、 (a) an oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is a DNA sequence of the 20th intron site DNA sequence of the human elastin gene; An oligonucleotide having a sequence containing the 17th DNA polymorphic site, or having the 3 'end of the oligonucleotide located one to several bases upstream from the polymorphic site,
(b) ヒト ·エラスチン遺伝子にハイブリダィズすることができる遺伝子多 型検出用プライマー又はプローブとしてのオリゴヌクレオチドであって、 該 オリゴヌクレオチドが、 ヒト ·エラスチン遺伝子の 23番目のイントロン部 位 DN A配列の 24番目の DN Aの遺伝子多型部位を含む配列を有するか、 又は、 該オリゴヌクレオチドの 3' 末端が該遺伝子多型部位よりも 1塩基か
ら数塩基上流に位置するものであるオリゴヌクレオチド。 (b) an oligonucleotide as a primer or a probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is a nucleotide of the 23rd intron DNA sequence of the 23rd intron of the human elastin gene; Has a sequence containing the polymorphic site of the third DNA, or whether the 3 ′ end of the oligonucleotide is one base more than the polymorphic site of the gene An oligonucleotide that is located a few bases upstream.
項 7. ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DN A配列の 17番目の DNAの遺伝子多型検出用プライマ一、 及び、 ヒト ·エラスチン 遺伝子の 23番目のイントロン部位 DNA配列の 24番目の DNAの遺伝子 多型検出用プライマーを含む脳動脈瘤の発症のリスク検出用診断キット。 項 8. ヒト ·エラスチン遺伝子の 20番目のイントロ 部位 DN A配列の 17番目の DN Aが野生型 （T) であって、 且つ 23 目のイントロン部位 DNA配列の 24番目の DNAが野生型 （T) から （C) に変異した変異型 である、 脳動脈瘤の発症のリスク検出のための DN A配列からなるハプロタ イブ及びその相補鎖。 以下、 本明細書におけるアミノ酸、 ペプチド、 塩基配列、 核酸等の略号に よる表示は、 I UPAC— I UBの規定 〔IUPAC- IUB communication on Biological Nomenclature, Eur. J. Biochem. , 138: 9 (1984)〕 、 「塩基配 列又はアミノ酸配列を含む明細書等の作成のためのガイドライン」 （特許庁 編） 及び当該分野における慣用記号に従うものとする。 Item 7. Intron region at position 20 of human elastin gene Primer for detecting a polymorphism at DNA position 17 of DNA sequence and intron region 23 of DNA sequence of human elastin gene at position 23 A diagnostic kit for detecting the risk of developing cerebral aneurysms, comprising a primer for detecting a gene polymorphism. Item 8. In the human elastin gene, the 17th DNA in the 20th intron DNA sequence is wild type (T), and the 24th DNA in the 23rd intron DNA sequence is wild type (T). A haplotype comprising a DNA sequence for detecting the risk of developing a cerebral aneurysm and a complementary strand thereof, which is a mutant from () to (C). Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in the present specification are referred to as I UPAC-I UB [IUPAC- IUB communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984) )], “Guidelines for Preparation of Descriptions Containing Base Sequences or Amino Acid Sequences” (edited by the Patent Office) and conventional symbols in the relevant field.
本発明において、 「遺伝子多型」 とは、 遺伝子中のある部位において、 ヒ トによって 2種以上の塩基が存在することをいい、 ここでは一般集団で頻度 の高い型を野生型、 それとは異なる頻度の低い塩基を有するものを変異型と いう。 In the present invention, the term "gene polymorphism" refers to the presence of two or more bases in a certain site in a gene depending on the human.Here, the type frequently used in the general population is different from the wild type. Those having infrequent bases are called mutants.
また、 ハプロタイプとは、 一つのアレル （ハプロイド） に存在する遺伝子 変異の組み合わせをいう。 A haplotype refers to a combination of gene mutations present in one allele (haploid).
本発明八プロタイプの具体例としては、 後述する実施例に示される、 イン トロン 20/イントロン 23八プロタイプと名づけられた、 ヒト ·エラスチ ン遺伝子の 20番目のイントロン部位 DN A配列の 17番目の DN Aが野生 型であって、 且つ 23番目のイントロン部位 DN A配列の 24番目の DN A が Tから Cに変異した変異型からなるハプロタイプを挙げることができる。 図 4に示すように、 ヒト 'エラスチン遺伝子は、 インデック（Indik)らによ つて、 全長 55 k bの 34個のェキソンからなる遺伝子であると報告されて
いる（Bashir, M., et al.， J. Biol. C em. , 264， 8887-8891 (1989)) 0 本発明者は、 104対の脳動脈瘤の罹患同胞対 （脳動脈瘤患者同胞） にお いて、 遺伝要因の強さ（As= 6)を考慮したシュミレーシヨンにより、 この同 胞対数で十分遺伝子座を特定でき得ると予想し、 罹患同胞対連鎖解析法によ り、 連鎖検定を行った。 ゲノム全域での連鎖解析の結果、 7番染色体のマー カー D 7 S 2472が最も強い連鎖結果を得た。 多点連鎖解析の結果、 この 領域で最大 l o d=3. 22を得た。 この結果から、 脳動脈瘤遺伝子座のひ とつが 7 Q 1 1. 23に特定され、 この領域に疾患遺伝子が存在することが 強く示唆された。 Specific examples of the eight prototypes of the present invention include the 17th intron site DNA sequence of the 20th intron site of the human elastin gene, named intron 20 / intron 238 protypes, which will be described in Examples described later. Is a wild-type, and a haplotype consisting of a mutant in which the 24th DNA of the 23rd intron site DNA sequence is mutated from T to C. As shown in Figure 4, the human 'elastin gene was reported by Indik et al. To be a 55 kb-long gene consisting of 34 exons. (Bashir, M., et al., J. Biol. Cem., 264, 8887-8891 (1989)) 0 The present inventor considered 104 pairs of sibs with cerebral aneurysms ), It is expected that simulation considering the strength of genetic factors (As = 6) will allow sufficient identification of the locus with this sibling logarithm. Was done. As a result of linkage analysis across the genome, the marker D7S2472 on chromosome 7 obtained the strongest linkage result. As a result of multipoint linkage analysis, a maximum lod = 3.22 was obtained in this region. From these results, one of the cerebral aneurysm loci was identified at 7Q11.23, and it was strongly suggested that disease genes exist in this region.
本発明者は、 最も強い連鎖を認めた 7 q 1 1. 23領域の遺伝マーカ一に 隣接してエラスチン遺伝子が存在していることを見出し、 該遺伝子について、 遺伝子変異のスクリーニングを直接シーケンス解析 （実施例 3参照） にて行 なった。 その結果、 13ケ所で一塩基多型を検出した。 さらに本発明者は、 エラスチン遺伝子多型と脳動脈瘤との関連性を調べるため、 すべての多型に ついてアレル頻度の比較をケースコントロールスタディで行った。 個々の変 異では有意な差を得るにいたらなかったが、 2つの多型を組み合わせてハプ 口タイプ解析をおこなった結果、 ヒト ·エラスチン遺伝子のイントロン 20 の 17番目の DNAが野生型 T (チミン） で、 イントロン 23の 24番目の D N Aが C (シトシン）に変異したハプロタイプで、 もっとも強い有意差を得、 この両者の変異の組み合わせが、 脳動脈瘤疾患に関与していることを見出し た。 The present inventor found that the elastin gene was present adjacent to the genetic marker in the 7q1.1.23 region where the strongest linkage was recognized, and conducted direct sequence analysis for gene mutation screening for the gene. (See Example 3). As a result, single nucleotide polymorphisms were detected at 13 sites. Furthermore, the present inventor conducted a case control study to compare allele frequencies for all polymorphisms in order to examine the association between the elastin gene polymorphism and cerebral aneurysms. Although individual mutations did not result in significant differences, haplotype analysis of the combination of the two polymorphisms revealed that the 17th DNA of intron 20 of the human elastin gene had wild-type T (thymine The haplotype in which the 24th DNA of intron 23 was mutated to C (cytosine) showed the strongest significant difference, and found that the combination of both mutations was involved in cerebral aneurysm disease.
本発明において遺伝子は、 2本鎖 DN Aのみならず、 それを構成するセン ス鎖およびアンチセンス鎖といった各 1本鎖 DN Aを包含する。 またその長 さに何ら制限されるものではない。 従って、 本発明の遺伝子 （DNA) には、 特に言及しない限り、 ヒトゲノム DNAを含む 2本鎖 DNA、 および 1本鎖 DNA (センス鎖） 、 並びに該センス鎖と相補的な配列を有する 1本鎖 DN A、 及びそれらの断片のいずれもが含まれる。 また、 本発明中、 遺伝子 (D NA) には、 調節領域、 コード領域、 ェキソン、 イントロンが含まれる。 ま た、 ポリヌクレオチドには、 RNA、 DNAのいずれもが含まれ、 ゲノム D
NA、 合成 D NAも含まれる。 In the present invention, the gene includes not only a double-stranded DNA, but also a single-stranded DNA such as a sense strand and an antisense strand constituting the double-stranded DNA. It is not limited by its length. Therefore, unless otherwise specified, the gene (DNA) of the present invention includes double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand), and single-stranded DNA having a sequence complementary to the sense strand. DNA and any of their fragments are included. In the present invention, the gene (DNA) includes a regulatory region, a coding region, an exon, and an intron. In addition, polynucleotides include both RNA and DNA, and genomic DNA NA and synthetic DNA are also included.
以上のとおり、 本発明遺伝子の八プロタイプおよびその検出方法の提供は、 脳動脈瘤のリスクの解明、 把握、 診断、 予防および治療等に極めて有用な情 報乃至手段を与える。 また、 本発明遺伝子の八プロタイプは、 例えば、 野生 型に改変する新規薬剤の開発の上でも好適に利用できる。 更に、 個体或は組 織における本発明遺伝子の八プロタイプの検出は、 上記脳動脈瘤の解明や診 おいても好適に利用できる。 As described above, the provision of the eight prototypes 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. Further, the eight prototypes of the gene of the present invention can be suitably used, for example, in the development of a novel drug that changes to a wild type. Further, the detection of the eight prototypes of the gene of the present invention in an individual or a tissue can be suitably used in elucidation and diagnosis of the cerebral aneurysm.
本発明の遺伝子又は D N Aの合成は、 ホスホルアミダイト法またはトリエ ステル法による化学合成によることもでき、 市販されている自動オリゴヌク レオチド合成装置で行なうこともできる。 二本鎖断片は、 相補鎖を合成し、 適当な条件下で該鎖を共にァニ一リングさせるか、 または適当なプライマ一 配列と D N Aポリメラーゼを用いて相補鎖を付加するかによって、 化学合成 した一本鎖生成物から得ることもできる。 本発明の八プロタイプ The gene or DNA of the present invention can be synthesized by chemical synthesis using the phosphoramidite method or the triester method, or can be performed using a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments are chemically synthesized by synthesizing a complementary strand and either annealing the strands together under appropriate conditions or adding a complementary strand using an appropriate primer sequence and DNA polymerase. It can also be obtained from single stranded products. Eight prototypes of the present invention
本発明遺伝子の八プロタイプの具体的態様としては、 例えば、 上記ヒト · エラスチン遺伝子の配列番号 1に示される 2 0番目のイントロン部位 D N A 配列の 1 7番目の D N Aが Tであって、 且つ配列番号 2に示される 2 3番目 のイントロン部位 D N A配列の 2 4番目の D N Aが Tから Cに変異した D N A配列からなるハプロタイプが例示される。 As a specific embodiment of the eight prototypes of the gene 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 the sequence A haplotype consisting of a DNA sequence in which the 24th DNA of the 23rd intron site DNA sequence shown in No. 2 is mutated from T to C is exemplified.
また、 本発明のハプロタイプは、 上記ヒト ·エラスチン遺伝子の 2 0番目 のイントロン部位 D N A配列の 1 7番目の D N Aが Tであって、 且つ 2 3番 目のイントロン部位 D N A配列の 2 4番目の D N Aが Tから Cに変異した D N A配列からなる配列のハプロタイプの相補鎖を有する D N A配列からなる ものも包含する。 In the haplotype of the present invention, the 17th DNA of the 20th intron site DNA sequence of the human elastin gene is T, and the 24th DNA of the 23rd intron site DNA sequence Also includes a DNA sequence having a haplotype complementary strand to a sequence comprising a DNA sequence mutated from T to C.
本発明の上記ハプロタイプを含む遺伝子は、 全長遺伝子であってもよいし、 そのハプロタイプを含む部分遺伝子であってもよい。 The gene containing the haplotype of the present invention may be a full-length gene or a partial gene containing the haplotype.
本発明遺伝子は、 本発明により開示された本発明遺伝子の具体例の配列情 報に基づいて、 一般的遺伝子工学的手法により容易に製造 ·取得することが
できる C Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989) ；続生化学実験講座 「遺伝子研究法 I、 II、 III」 、 日本生化学会編 (1986) など参照〕 The gene of the present invention can be easily produced and obtained by general genetic engineering techniques based on the sequence information of specific examples of the gene of the present invention disclosed by the present invention. C Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Seminar on Seismic Chemistry "Gene Research Methods I, II, III", edited by Biochemical Society of Japan (1986) etc.)
具体的には、 本発明遺伝子の八プロタイプを有する、 例えば、 配列番号 1 及び 2の部分を有する、 適当な起源より、 常法に従ってゲノム DNAライブ ラリーを調製し、 該ライブラリ一から、 本発明遺伝子に特有の変異を含む適 当なプロ一ブを用い、 所望のクローンを選択することによって実施できる。 上記において、 ゲノム DNAの起源としては、 本発明の遺伝子を有する各 種の細胞、 組織やこれらに由来する培養細胞などが例示される。 具体的には、 血液、 唾液、 リンパ液、 気道粘液、 尿又は精液などの体液などを例示するこ とができる。 血液としては、 血清、 血漿などが挙げられる。 Specifically, a genomic DNA library having eight prototypes of the gene of the present invention, for example, having portions of SEQ ID NOs: 1 and 2, is prepared from an appropriate source according to a conventional method, This can be performed by selecting a desired clone using an appropriate probe containing a mutation specific to the gene. In the above, 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 blood, saliva, lymph, airway mucus, and bodily fluids such as urine and semen. Blood includes serum, plasma and the like.
本発明の遺伝子をゲノム DN Aライブラリーからスクリーニングする方法 も、 特に制限されず、 通常の方法で行なうことができる。 例えば、 目的の D N A配列に選択的に結合するプローブを用いたプラークハイプリダイゼ一シ ヨン、 コロニーハイブリダィゼーシヨン又はこれらの組み合わせなどが例示 できる。 The method for screening the gene of the present invention from a genomic DNA library is not particularly limited, and can be performed by a usual method. For example, plaque hybridization, colony hybridization, or a combination thereof using a probe that selectively binds to a target DNA sequence can be exemplified.
さらに、 抽出した遺伝子を遺伝子増幅法によって増幅することにより、 ス クリーニングをより容易にかつ精度の高いものにすることができる.。 遺伝子 増幅法の例としては、 PCR法 (Saiki,R. K. , Bugawan, T. L. , et al.， Nature, 324, 163-166 (1986) ) 、 N A S B A法 （ Co即 tom， J.， Nature, 650， 9卜 92 (1991) ) 、 TMA法 ( Kacian, D. L. , and Fultz,T. J. ，米国特許番号 5, 399,491 (1995)) および SDA法 (Walker, G.T., Little, M.C., et al, Proc. Natl. Acad. Sci. USA, 89, 392-396(1992)) などが挙げられる。 Furthermore, 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)) and the NASBA method (Co Immediate Tom, J., Nature, 650, 9). Uto 92 (1991)), TMA method (Kacian, DL, and Fultz, TJ, U.S. Patent No. 5,399,491 (1995)) and SDA method (Walker, GT, Little, MC, et al, Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992)).
本発明の遺伝子八プロタイプの取得に際しては、 PCR法 [Science, 230, 1350 (1985)3 による D N A増幅法が好適に利用できる （Saiki, R. K. , Bugawan, T. L.， et al. , Nature, 324, 163-166 (1986)) 。 In obtaining the eight gene prototypes of the present invention, a DNA amplification method based on the PCR method [Science, 230, 1350 (1985) 3] can be suitably used (Saiki, RK, Bugawan, TL, et al., Nature, 324, 163-166 (1986)).
かかる遺伝子増幅法の採用に際して使用されるプライマ一は、 本発明によ つて明らかにされた本発明の遺伝子の配列情報に基づいて適宜設定でき、 常 法に従って合成できる。 また、 増幅させた DN A断片の単離精製は、 前記の
通り常法に従うことができ、 例えば、 ゲル電気泳動法や、 カラムにて精製す る方法などを用いることができる。 或いは、 マススペクトルで観察すること もできる。 The primer 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 according to the present invention, and can be synthesized according to a conventional method. Further, the isolation and purification of the amplified DNA fragment is performed as described above. Conventional methods can be followed, for example, gel electrophoresis, column purification, and the like. Alternatively, it can be observed with a mass spectrum.
上記で得られる本発明遺伝子八プロタイプは、 例えば、 一塩基プライマー 伸長法を用いて、 また、 その DNA断片は、 常法、 例えばジデォキシ法 CProc. Nail. Acad. Sc USA., 74, 5463 (1977)3 やマキサム一ギルバー ト法 [Methods in Enzymology, 65, 499 (1980)] 、 或いは、 市販のシ一クェ ンスキットなどを用いて、 その塩基配列を決定することができる。 The eight genes of the gene of the present invention obtained above can be obtained, for example, by using a single-base primer extension method, and the DNA fragment thereof can be obtained by a conventional method, for example, a dideoxy method CProc. Nail. Acad. Sc USA., 74, 5463 ( 1977) 3 or the Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)], or a commercially available sequence kit can be used to determine the nucleotide sequence.
このようにして得られる本発明の遺伝子 （ハプロタイプ） を用いることに より、 例えば、 該遺伝子の一部又は全部の塩基配列を利用することにより、 個体もしくは各種組織における本発明遺伝子の有無を特異的に検出すること ができる。 本発明の判定方法 By using the gene (haplotype) of the present invention obtained in this way, for example, by utilizing a part or the entire nucleotide sequence of the gene, the presence or absence of the gene of the present invention in an individual or various tissues can be specifically determined. Can be detected. Determination method of the present invention
本発明は、 ヒト ·エラスチン遺伝子の遺伝子多型を同定することにより、 脳動脈瘤の発症のリスクの存在を判定する方法に関する。 具体的には、 本発 明の方法は、 該遺伝子のィントロン部分の遺伝子多型を同定することによつ て判定するものである。 詳しくは、 上述のごとく、 20番目のイントロン部 位 （以下、 「イントロン 20」 という場合がある。 ） の 1 7番目の遺伝子多 型及び 2 3番目のイントロン部位 （以下、 「イントロン 23」 という場合が ある。 ） の 24番目の遺伝子多型を同定することによって、 脳動脈瘤の発症 リスクの存在を判定する。 The present invention relates to a method for determining the presence of a 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 genetic polymorphism in the intron portion of the gene. Specifically, as described above, the 17th polymorphism at the 20th intron site (hereinafter sometimes referred to as “intron 20”) and the 23rd intron site (hereinafter referred to as “intron 23”) Identify the 24th polymorphism in) to determine the risk of developing a cerebral aneurysm.
該 2力所の遺伝子多型を同定することによつて脳動脈瘤の発症リスクの存 在を判定する方法としては、 検出すべき遺伝子の遺伝子型が同定される方法 であれば、 特に限定されないが， 例えば、 以下の方法が挙げられる。 The method of determining the risk of developing a cerebral aneurysm by identifying the two polymorphisms is not particularly limited as long as the genotype of the gene to be detected is identified. However, for example, the following method can be used.
(1) 検体からヒ卜 ·エラスチンをコードする遺伝子 （ヒ卜 ·エラスチン 遺伝子） を含む核酸 （DNA) を得る方法。 該 DNAは、 ヒト DNAの全長 DNAであってもよいが、 少なくとも上記本発明のかかる遺伝子の遺伝子多 型位置を含む部分 DN Aであれば、 特に限定されない。
また、 該 D N Aは、 ヒトに.由来するものであれば、 特に制限されず、 ヒト D NAを含む血液、 生体材料組織等の生体試料から採取したものを用いるこ とができる。 これら試料から D N Aを抽出する方法は、 常法に従って行えば よく、 例えば、 市販のキットや装置を使用することができる （QIAGEN Blood & Cel l Cul ture D NA ki t (QIAGEN社製)） 。 (1) A method for obtaining a nucleic acid (DNA) containing a gene encoding human elastin (human elastin gene) from a specimen. The DNA may be full-length DNA of human DNA, but is not particularly limited as long as it is at least a partial DNA containing the polymorphic position of the gene of the present invention. The DNA is not particularly limited as long as it is derived from humans, and DNA collected from a biological sample containing human DNA, such as blood or a biological material tissue, can be used. DNA may be extracted from these samples by a conventional method, for example, using a commercially available kit or device (QIAGEN Blood & Cell Culture DNA Kit (manufactured by QIAGEN)).
( 2 ) 本発明の同定方法 (2) Identification method of the present invention
上記 （1 ) で得られた D NAの量が微量の場合には、 以下に示す検出方法 の前に、 上記に記載したような遺伝子増幅法によって増幅することにより、 同定をより容易にかつ精度の高いものにすることができる。 上記方法および 以下に示す各方法において、 検体としての遺伝子増幅法で増幅させる D N A 断片は、 前述した変異の存在が想定される特定部位の少なくとも 1つを含む 限り、 特に限定されるものではないが、 通常、 約 5 0から数千塩基の長さ、 好ましくは 5 0から数百塩基の長さを有するものがよい。 特に遺伝子多型部 位の全てを含むものが好ましい。 If the amount of DNA obtained in (1) above is very small, amplification can be performed by the gene amplification method as described above before the detection method shown below to make identification easier and more accurate. Can be high. In the above method and each of the methods described below, 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 of the specific sites where the above-mentioned mutation is assumed to be present. It usually has a length of about 50 to several thousand bases, preferably 50 to several hundred bases. In particular, those containing all of the polymorphism sites are preferred.
以下の検出方法で用いられるプロ一ブまたはプライマーとしては、 本発明 の遺伝子ハプロタイプの D N A塩基配列に関する情報をもとにして変異を含 む部分を化学合成した D N Aなどが一般に使用できる。 また、 既に取得され た本発明遺伝子八プロタイプやその変異塩基を含む D N A断片も良好に利用 できる。 また、 本発明遺伝子八プロタイプの塩基配列情報に基づき設定した プライマーをスクリーニング用プローブとして用いることもできる。 また、 遺伝子多型部位を含まなくとも、 ヒト ·エラスチン遺伝子のセンス鎖又はァ ンチセンス鎖にハイブリダィズでき、 以下の検出方法に利用できるものであ れば特に限定されない。 As probes or primers used in the following detection methods, DNA obtained by chemically synthesizing a portion containing a mutation based on information on the DNA base sequence of the gene haplotype of the present invention can be generally used. In addition, a DNA fragment containing the eight prototypes of the gene of the present invention and its mutant bases, which have already been obtained, can also be used favorably. Further, a primer set based on the base sequence information of the eight prototypes of the gene of the present invention can be used as a screening probe. In addition, there is no particular limitation as long as it can hybridize to the sense strand or antisense strand of the human elastin gene without containing the gene polymorphism site and can be used for the following detection methods.
より具体的には、 前記プローブ又はプライマーとして用いられるヌクレオ チド配列は、 ヒト ·エラスチン遺伝子のイントロン 2 0の D N A配列の 1 7 番目の核酸、 イントロン 2 3の D N A配列の 2 4番目の核酸を検出できるよ うに設定された配列に対応する部分ヌクレオチド配列であって、 少なくとも 1 5個の連続した塩基、 好ましくは少なくとも 2 0個の連続した塩基、 より 好ましくは 3 0個の連続した塩基、 最も好ましくは 5 0個の連続した塩基を
有するものが挙げられる。 或いは、 前記配列を有する陽性クローンそれ自体 をプローブとして用いることも出来る。 More specifically, the nucleotide sequence used as the probe or primer detects 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. A partial nucleotide sequence corresponding to the sequence set as possible, comprising at least 15 contiguous bases, preferably at least 20 contiguous bases, more preferably 30 contiguous bases, most preferably Produces 50 consecutive bases Ones having. Alternatively, the positive clone itself having the above sequence can be used as a probe.
1) ヌクレオチド直接塩基決定法 1) Direct nucleotide base determination method
まず第一に、 本発明に係る遺伝子の検出は、 この種の遺伝子の塩基配列決 定に慣用されている、 例えばダイデォキシ法（Sanger, et al.， Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977))、 マキサム一ギルバ一 ト法 (Methods in Enzymology, 65, 499 (1980)) などの直接塩基配列決定法に従 つて実施することができる。 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, by the dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci. USA, 74). , 5463-5467 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499 (1980)).
また、 これらの方法と上述の P CR法などの DN A増幅法を組み合わせた 方法に従って実施することもできる。 特に、 少量の DN A試料を用いて簡便 かつ容易にしかも感度および精度の高い検出を可能とする観点から、 PCR 法もしくはそれに準じた DNA増幅法を組み合わせて行なうことが好ましい この好ましい方法は、 基本的には、 例えば PC R法で増幅させた遺伝子断 片 （検体） をプラスミドにクローニングし、 次いでダイデォキシ法、 マキサ ムーギルバ一ト法などに従って、 直接塩基配列をシーケンスすることにより、 また簡便には市販のシークェンスキットなどを用いてヌクレオチド配列を決 定することにより実施できる。 かくして、 ヒト ·エラスチン遺伝子のイント ロン 2 0および 23における特定の遺伝子多型部位の変異の存在を決定でき、 またそのハプロタイプを決定できる。 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, it is preferable to carry out PCR in combination with a DNA amplification method equivalent thereto. Specifically, for example, a gene fragment (sample) amplified by the PCR method is cloned into a plasmid, and then the base sequence is directly sequenced according to the dideoxy method, the Maxa-Mougill-But method, etc. It can be performed by determining the nucleotide sequence using a sequence 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 ) 対立遺伝子特異的ォリゴヌクレオチド一ドットプロット分析 2) Allele-specific oligo nucleotide single-dot plot analysis
本発明に係る検出方法の別法としては、 対立遺伝子特異的オリゴヌクレオ チド（AS0) ドットプロッ ト法（Conner, B. J., et al.， Proc. Natl. Acad. Sci., 80, 278-282 (1983)) に従った方法などが挙げられる。 該方法は、 例え ば、 目的とする一塩基多型部位を挟むように設計したフォワード ·プライマ —およびリバース ·プライマ一を利用して PCR増幅した遺伝子断片に対す る対立遺伝子特異的オリゴヌクレオチド ·プローブにハイブリダィズする D NA靳片を、 ドット ·プロット分析することにより実施できる。 かくして、 該 DN A断片中に一塩基多型が存在するか否かを決定することができる。 As an alternative to the detection method according to the present invention, an allele-specific oligonucleotide (AS0) dot plot method (Conner, BJ, et al., Proc. Natl. Acad. Sci., 80, 278-282 ( 1983)). The method includes, for example, an allele-specific oligonucleotide probe for a gene fragment PCR-amplified using a forward primer designed to sandwich a target single nucleotide polymorphism site and a reverse primer. The DNA fragments that hybridize to DNA can be analyzed by dot plot analysis. Thus, it is possible to determine whether a single nucleotide polymorphism is present in the DNA fragment.
3) 一塩基プライマー伸長法
本発明エラスチン遺伝子多型 ひ、プロタイプ） の検出は、 スナップショッ ト法 (Kuppuswamy, M. N. , et al. Proc. Natl. Acad. Sci. USA 88, 1143- 1147(1991) ) 、 ピロシーケンス法 （Ronaghi, Μ·， et al. , Science, 281, 363-365 (1998)) 、 特開 2000- 279197号に開示の点変異検出法のような一塩基 プライマー伸長法を用いて実施することもできる。 これらの場合、 目的とす る遺伝子多型部位の直前の塩基または数塩基前の塩基に対応するように設定 したプライマー、 即ち、 その 3' 末端を目的とする変異の 1塩基上流または 近傍に設定したプライマーを DN A検体にァニーリングさせて実施すること もできる。 上記各方法は、 市販の SNP s (—塩基多型） 検出用キットおよ び該キットに添付のソフトウエアを利用して実施することができる。 3) Single nucleotide primer extension method The elastin gene polymorphism (prototype) of the present invention can be detected by a snapshot method (Kuppuswamy, MN, et al. Proc. Natl. Acad. Sci. USA 88, 1143-1147 (1991)), a pyrosequencing method ( Ronaghi, Μ ·, et al., Science, 281, 363-365 (1998)), and a single-base primer extension method such as the point mutation detection method disclosed in JP-A-2000-279197. . In these cases, a primer set so as to correspond to the base immediately before or several bases before the target polymorphic site, that is, the 3 'end is set one base upstream or close to the target mutation. The primers can also be annealed to the DNA sample. Each of the above methods can be carried out using a commercially available kit for detecting SNP s (—base polymorphism) and software attached to the kit.
例えばスナップショット法は、 AB I PR I SM SNaP s ho t d dNTP P r ime r E t en s i on K i t (AB Iバイオシステム ズ社製）を用いて実施できる。 一塩基多型は、 反応後に生成した蛍光フラグメ ントを、 AB I PR I SM310/337/3100/3700 DNA A n a 1 y z e r (いずれも A B Iバイオシステムズ社製）と Ge ne S c an ソフトウェアを用いて検出 ·解析することもできる。 For example, the snapshot method can be carried out using ABI PRIMSMNaPshot dNTPPrimerEntenSionKit (manufactured by ABI Biosystems). For single nucleotide polymorphism, the fluorescent fragments generated after the reaction are analyzed using AB IPRI SM310 / 337/3100/3700 DNA Analyzer (both manufactured by ABI Biosystems) and GeneScan software. Detection and analysis are also possible.
ピロシーケンス法は、 例えば、 以下のごとくして実施できる。 即ち、 血液 サンプルなどから常法によりゲノム DN Aを単離し、 ピオチン標識したブラ イマ一を用いて遺伝子多型部位を含む数十から数百塩基を PC R増幅させ、 マグネットビーズを用いて一本鎖 DNAを精製し、 この精製 DNAを検体と する。 該検体に、 所望の変異の数塩基上流からシーケンスするように設定し たプライマ一をァ二一リングさせ、 次いでソフトウェアに入力された変異付 近のシーケンスに従って装置に 1種類ずつ dNTPを添加する。 DNAポリ メラ一ゼが塩基伸長するとピロリン酸（PP i)を生成するので、 該 PP iを スルフリラーゼ（Sulfurylase)により ATPに返還させ、 これをルシフェラー ゼの基質として、 化学発光を発光検出器、 CCDカメラなどを用いて検出す る。 添加した ci N T Pに応じて得られる発先のピークを解析することによつ て遺伝子のタイピングが可能となる。 該方法を用いれば、 96サンプルを 1 5分ほどでタイピングすることができる。
上記方法において用いる試薬および装置としては、 通常のもの、 例えば D NAポリメラーゼ、 ATP-スルフリラーゼ、 ルシフェラ一ゼおよびアビラ一 ゼ（apyrase)の 4種の酵素混合液、 ルシフェリンおよび AP S (アデノシン 5' 硫酸リン酸） からなる基質液、 d AT P (デォキシアデノシン α—チォ - 3リン酸）、 dCTP、 dGTPおよび dTTPからなる dNTPを構成要素 とする市販の SNP Re a g e n t K i t s (Pyrosequencing AB社製）な どの試薬、 自動 DNA配列分析のための PSQ 96システム（Pyrosequencing AB社製）及びその使用のための S N Pソフトウェア（Py r 0 s equenc i ng AB社製） などを用いることができる。 The pyrosequencing method can be performed, for example, as follows. In other words, genomic DNA is isolated from a blood sample or the like by a conventional method, PCR-amplified tens to hundreds of bases including the polymorphic site using a biotin-labeled primer, and used to separate the DNA using magnet beads. The strand DNA is purified, and the purified DNA is used as a sample. A primer set to sequence from a few bases upstream of the desired mutation is allowed to ring with the sample, and then dNTPs are added to the device one by one according to the sequence near the mutation input to the software. When DNA polymerase undergoes base extension, pyrophosphate (PPi) is generated. The PPi is returned to ATP by sulfurylase, and this is used as a substrate for luciferase. Detect using a camera. Gene typing can be performed by analyzing the peak of the destination obtained according to the added ci NTP. Using this method, 96 samples can be typed in about 15 minutes. As the reagents and equipment used in the above method, conventional reagents such as DNA polymerase, ATP-sulfurylase, a mixture of four kinds of enzymes of luciferase and apyrase, luciferin and APS (adenosine 5 'sulfate) Phosphoric acid), a commercially available SNP Reagent K its (manufactured by Pyrosequencing AB) comprising dATP (doxyadenosine α-thio-3phosphate), dCTP, dGTP and dNTP consisting of dTTP ), A PSQ 96 system for automated DNA sequence analysis (manufactured by Pyrosequencing AB), and SNP software for use thereof (PyroSequencing AB) can be used.
また、 上記ピロシーケンス法は、 例えば米国特許第 6， 159， 693号 の記載に従って、 核酸を単離後、 増幅し、 増幅した PC R産物を精製後、 R EAD I T™ S y s t em (プロメガ ·コーポレ一ション社製）を用いて、 こ れにピロリン酸を反応させ、 得られたデ一夕を分析することによつても実施 できる。 このデ一夕分析には、 例えば市販の READ I T技術（プロメガ ·コ 一ポレーシヨン社製）を利用した Ex c e 1分析を採用できる。 In addition, the above pyrosequencing method involves isolating and amplifying a nucleic acid and purifying the amplified PCR product according to the description in, for example, US Pat. No. 6,159,693, and reading the amplified PCR product. The reaction can also be carried out by reacting this with pyrophosphoric acid and analyzing the resulting residue. For this overnight analysis, for example, an Exce1 analysis using commercially available READ IT technology (promega Co., Ltd.) can be employed.
4) PCR一単鎖高次構造多型 (SSCP)分析法 4) PCR single-stranded higher-order polymorphism (SSCP) analysis method
更に、 本発明に係る検出法には、 前述した PCR増幅産物 (一本鎖 DNA) を非変性ポリアクリルアミドゲル電気泳動して、 その移動度の差異により一 塩基変異の有無を識別する PC R— S S CP法 （Orita, M. , Iwahara, Η. , et al.， Proc. Natl. Acad. Sci. USA, 86, 2776-2770(1989) , Orita, et al., Genomics, 5, 874-879 (1989)) などを採用することもできる。 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, and PCR is used to discriminate the presence or absence of a single nucleotide mutation based on the difference in mobility. SSCP method (Orita, M., Iwahara, II., Et al., Proc. Natl. Acad. Sci. USA, 86, 2776-2770 (1989), Orita, et al., Genomics, 5, 874-879 (1989)).
5) PCR—制限酵素断片長多型 (RFLP) 分析法 5) PCR—restriction fragment length polymorphism (RFLP) analysis method
本発明のエラスチン遺伝子の遺伝子多型または八プロタイプの検出にあた り、 検出目的とする変異を含む核酸配列が制限酵素認識部位を含んでいる場 合には、 制限酵素断片長多型分析法 （RFLP 法： Botstein, D. R. , et al., Am. J. Hum. Gen., 32, 314-331 (1980)) を用いてもよい。 In the detection of the elastin gene polymorphism or octaprotype of the present invention, when the nucleic acid sequence containing the mutation to be detected contains a restriction enzyme recognition site, restriction enzyme fragment length polymorphism analysis is performed. The method (RFLP method: Botstein, DR, et al., Am. J. Hum. Gen., 32, 314-331 (1980)) may be used.
具体的には、 エラスチン遺伝子のイントロン 20の 17番目の核酸配列が 野生型 （T) であるか変異型 （C) であるかどうか、 17番目の核酸が野生 型 （T) であるか変異型 （C) であるかを検出するため、 若しくはイントロ
ン 23の 24番目の核酸配列が野生型か変異型であるかどうか、 24番目の 核酸が Tであるか Cであるかを認識するための、 それぞれの変異箇所の前後 配列を認、識し得る制限酵素を用いればよい。 Specifically, whether the 17th nucleic acid sequence of intron 20 of the elastin gene is wild-type (T) or mutant (C), and whether the 17th nucleic acid is wild-type (T) or mutant (C) to determine if Identify the sequence before and after each mutation to identify whether the 24th nucleic acid sequence of protein 23 is wild-type or mutant, and whether the 24th nucleic acid is T or C. The obtained restriction enzyme may be used.
かかる RF LP法に用いられる酵素としては、 目的とするそれぞれの変異 箇所の前後配列を認識し得る各種公知の制限酵素を用いることができる。 As the enzyme used in the RFLP method, various known restriction enzymes capable of recognizing the sequence before and after each target mutation site can be used.
該 RFLP法は、 より好適には、 PCR— RFLP法、 即ち、 予め PCR 法またはその変法などによって検体 DNAを増幅 ·調製後、 多量に調製され 且つ濃縮された検体 DN Aについて RF LP法を実施する方法を用いられる。 かくして、 特異的切断サイ卜の存在の有無を検出することができる。 More preferably, the RFLP method is a PCR-RFLP method, that is, after amplifying and preparing a sample DNA by a PCR method or a modified method thereof in advance, the RFLP method is performed on a large amount of a prepared and concentrated sample DNA. The method of implementation is used. Thus, the presence or absence of a specific cleavage site can be detected.
P CR— RFLP法による本発明エラスチン遺伝子の八プロタイプの検出 は、 より具体的には、 例えば、 次の方法に従って行われる。 即ち、 まず、 ヒ ト生体試料からエラスチン遺伝子の D N Aを抽出し、 該遺伝子のィントロン 20及び または 23の遺伝子多型部位を含む領域の DN A断片を増幅し、 多量に且つ濃縮された検体サンプルを得る。 次いで、 増幅 DNA検体を特定 の制限酵素 （即ち、 野生型又は変異型のいずれか一方のみを消化できる酵 素） を用いて消化し、 DNAの切断様式 （切断の有無、 切断フラグメントの 塩基長など） を常法に従って確認する。 The detection of the eight prototypes 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, the DNA of the elastin gene is extracted from a human biological sample, the DNA fragment of the region containing the gene polymorphism site of intron 20 and / or 23 of the gene is amplified, and a large and concentrated sample sample is obtained. obtain. Next, the amplified DNA sample is digested with a specific restriction enzyme (ie, an enzyme capable of digesting either the wild type or the mutant type only), and the DNA cleavage mode (such as presence or absence of cleavage, base length of the cleavage fragment, etc.) ) Is checked in accordance with the usual method.
本発明エラスチン遺伝子の遺伝子多型またはハプロタイプの検出は、 以下 に示すィンべ一ダ一（Invader)法および定量的リアルタイム P C R検出法 (TaqMan法）により実施することもできる。 Detection of the genetic polymorphism or haplotype of the elastin gene of the present invention can also be carried out by the following Invader method and the quantitative real-time PCR detection method (TaqMan method).
6) インベーダー法 6) Invader method
ィンベーダ一法の実施には、 以下の文献が参照できる。 The following documents can be referred to for the implementation of the invader-one method.
• Lyamichev, V. , et al., Nat. Biotechnol. , 17(3) 292-296(1999) および • Lyamichev, V., et al., Nat. Biotechnol., 17 (3) 292-296 (1999) and
·国際特許公開 TO98Z23774号（98/6/4)。 · International Patent Publication TO98Z23774 (98/6/4).
該方法は、 ゲノム DN Αの一塩基多型を分析するのに予め標的 DN Αを増 幅する必要がない方法で、 以下のように実施される。 The method is a method in which it is not necessary to amplify a target DN in advance to analyze a single nucleotide polymorphism in a genomic DN, and is carried out as follows.
目的とするエラスチン遺伝子のイントロン 20及び 23の特定部位に多型 が存在するかどうかを検出するために、 先ずゲノム DN Aを単離した後、 1
5から 50塩基長からなる 5 ' フラップと、 検出したい核酸を 5 ' フラップ の 3 ' 端に配し、 標的ゲノム DNAに相補するように合成された 30から数 百塩基のオリゴヌクレオチドからなる第一の標的プローブと、 検出したい核 酸に相補的な核酸を 3 ' 端に配する以外は、 標的ゲノム DNAに相補するよ うに合成された 15から数十塩基長のオリゴヌクレオチドからなるインべ一 ダー ·オリゴヌクレオチド ·プローブとを、 例えば自動合成機により合成す る。 これらのプローブと単離したゲノム DNAを適当な反応液中で反応させ た後、 変異核酸を含むインベーダー構造を認識し、 第一のプローブの 5 ' フ ラップを切断する酵素を添加する。 In order to detect whether a polymorphism exists at specific sites in introns 20 and 23 of the elastin gene of interest, first isolate genomic DNA, then The first consists of a 5 'flap of 5 to 50 bases in length and an oligonucleotide of 30 to several hundred bases synthesized to complement the target genomic DNA by placing the nucleic acid to be detected at the 3' end of the 5 'flap. Integrator consisting of 15 to several tens of nucleotides synthesized to complement target genomic DNA, except that the target probe and the nucleic acid complementary to the nucleic acid to be detected are placed at the 3 'end. · Oligonucleotides · Probes are synthesized by, for example, 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 cleaves the 5 ′ flap of the first probe is added.
これによつて， もし検体中のゲノム DNAが所望の検出目的の核酸を有し ている場合は、 前記制限酵素による切断によって、 検出目的の核酸を 3 ' 端 に有する 5 ' フラップが遊離し、 第一の反応が終了する。 もし、 検体中のゲ ノム DN Aが検出目的の配列を有していない場合は、 前記制限酵素による切 断が生じない。 As a result, if the genomic DNA in the sample contains the desired nucleic acid for detection, the 5 ′ flap having the nucleic acid for detection at the 3 ′ end is released by cleavage with the restriction enzyme, The first reaction ends. If the genomic DNA in the sample does not have the sequence to be detected, cleavage by the restriction enzyme does not occur.
制限酵素で切断された第一のプローブから遊離した 5' フラップは、 標的 として蛍光共鳴エネルギー移転 (FRET)プローブを使用する第二の反応におい て、 インベーダー'オリゴとして作用することによって反応を続ける。 The 5 'flap released from the first probe cleaved with the restriction enzyme continues by acting as an Invader' oligo in a second reaction using a fluorescence resonance energy transfer (FRET) probe as the target.
かくして、 第一のプローブ上の 5' フラップは、 3' 末端の塩基を除いて、 第二の反応に用いられる FRETプローブに相補的である。 Thus, the 5 'flap on the first probe, except for the base at the 3' end, is complementary to the FRET probe used in the second reaction.
次いで第二の反応に用いられる各 FRETプローブは、 検出される標的に かかわらず、 同一の配列を含んでいて、 本質的に 2つのエレメントからなる ように構築される。 即ち、 （1) 第一の反応から割裂した産物に相補する 3' 領域、 及び （2) —本鎖プローブを模倣するために複式を形成し、 そし て標的が共にハイブリダィズして、 それらがレポ一夕一蛍光色素とクェンチ ャ一蛍光色素を含んでいる自家相補的領域を有する。 Each FRET probe used in the second reaction is then constructed to contain the same sequence and consist essentially of two elements, regardless of the target to be detected. (1) a 3 'region complementary to the product cleaved from the first reaction, and (2) a duplex to mimic the stranded probe, and the targets hybridize together and become It has a self-complementary region containing an overnight fluorescent dye and a quencher fluorescent dye.
前記レポ一夕一蛍光色素は、 該レポーター蛍光色素が前記クェンチヤ一蛍 光色素と同一のプローブに結合している場合には蛍光共鳴エネルギー転移に よりその蛍光強度が抑制され、 前記クェンチヤ一蛍光色素と同一のプローブ に結合していない場合には蛍光強度が抑制されないものである。 したがって、
切断された第一のプロ一ブ*オリゴからの遊離した 5 ' フラップが、 F R E Tプローブにハイブリダィズしたとき、 それは第二の反応においてインべ一 ダー ·オリゴとして作用し、 制限酵素によって認識された侵入複合物を産生 する。 かくして、 F R E Tプローブの制限酵素による切断が、 二つの蛍光色 素を分離し、 検出可能な蛍光シグナルを産生する。 このようにして標準蛍光 マイクロタイタープレート読み取り機器が産物を読み取り検出することがで きる第一と第二の反応の組み合わせにより、 シグナルを 1から 1 X 1 0 6倍 まで増幅することができる。 本発明における、 所望の野生型又は変異型の核 酸の有無、 或いは、 ハプロタイプは、 インベーダー ·アツセィ法を用いるこ とによっても、 検出することができる。 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 quencher fluorescent dye is If the probe is not bound to the same probe, the fluorescence intensity is not suppressed. Therefore, When the free 5 'flap from the cleaved first probe * oligo hybridizes to the FRET probe, it acts as an invader oligo in the second reaction and the invasion recognized by the restriction enzyme Produce complex. Thus, restriction enzyme cleavage of the FRET probe separates the two fluorescent dyes and produces a detectable fluorescent signal. The combination of the first and second reactions, in which the standard fluorescence microtiter plate reader can read and detect the product, can amplify the signal from 1 to 1 × 10 6 fold. In the present invention, the presence or absence of a desired wild-type or mutant-type nucleic acid or a haplotype can also be detected by using the Invader-Attsey method.
7 ) 定量的リアルタイム P C R検出法 7) Quantitative real-time PCR detection method
本発明エラスチン遺伝子の遺伝子多型の検出は、 また定量的リアルタイム P C R検出法 (TaqMan法）によっても簡便に実施することができる。 The detection of the genetic polymorphism of the elastin gene of the present invention can also be easily performed by a quantitative real-time PCR detection method (TaqMan method).
該方法は、 以下のごとく実施できる。 即ち、 まず、 目的とする変異の有無 を検出する核酸部位を含む D N A断片を増幅するために、 約 1 5塩基ないし 約 3 0塩基からなるフォワード側プライマーとリバ一ス側プライマーを作成 する。 但し、 フォワード側プライマーとリバ一ス側プライマ一とも目的とす る変異の有無を検出する核酸部位を含まないように作成する。 次いで、 検出 用蛍光プローブとして、 1 5塩基ないし 5 0塩基からなる塩基配列を有する オリゴヌクレオチドにレポ一夕一蛍光色素とクェンチヤ一蛍光色素とが結合 されており、 且つフォワード側プライマーがハイブリダィズする領域とプロ ーブがハイプリダイズする領域が互いに重複することがない組み合わせを選 択したプローブを作成する。 該プローブは、 目的とする一塩基の核酸変異の 有無を検出するための対立遺伝子特異的配列に相補的な配列を有するように 作成する。 該プライマー及びプロ一ブを用いて、 検体中の測定すべきエラス チン遺伝子の所望の D NA断片領域を铸型として P C Rを行い、 反応液から 検出される蛍光をリアルタイムに測定する。 かくして、 変異の有無を検出す ることができる。 The method can be performed 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 bases to about 30 bases are prepared. However, both the forward primer and the reverse primer should be prepared so as not to include a nucleic acid site for detecting the presence or absence of a target mutation. Next, as a detection fluorescent probe, a region in which a repo overnight fluorescent dye and a quencher fluorescent dye are bound to an oligonucleotide having a base sequence consisting of 15 to 50 bases, and where the forward primer hybridizes Create a probe that selects a combination in which the region that the probe hybridizes with 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 nucleotide nucleic acid mutation. Using the primers and the probe, PCR is performed using the desired DNA fragment region of the elastin gene to be measured in the sample as type III, and the fluorescence detected from the reaction solution is measured in real time. Thus, the presence or absence of the mutation can be detected.
上記ィンべ一ダー ·アツセィ法や T a q M a n法に用いられるレポ一夕一
蛍光色素としては、 F AM (6-カルボキシ-フルォレツセイン）のようなフルォ レツセイン系蛍光色素が好ましく、 クェンチヤ一蛍光色素としては、 TAMRepo-One-Yuichi used in the above-mentioned invator-attsie method and TaqMan method As the fluorescent dye, a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable. As the quencher fluorescent dye, TAM
R A (6-力ルポキシ-テトラメチル-ローダミン）のような口一ダミン系蛍光色 素が好ましい。 これらの蛍光色素は公知であり、 市販のリアルタイム検出 P CR用キットに含まれているのでそれを用いることができる。 レポ一夕一蛍 光色素及びクェンチヤ一蛍光色素の結合位置は特に限定されないが、 通常、 プロ一ブのオリゴヌクレオチド部分の一端 （好ましくは 5， 末端） にレポ一 ター蛍光色素が、 他端にクェンチヤ一蛍光色素が結合される。 なお、 オリゴ ヌクレオチドに蛍光色素を結合する方法は公知であり、 例えば、 Noble et al., Nuc. Acids Res. 12:3387-3403 (1984) 及び Iyer et al.， J. Am. Chem.Mono-damine fluorescent dyes such as R A (6-potoxy-tetramethyl-rhodamine) are preferred. These fluorescent dyes are known and can be used because they are included in a commercially available kit for real-time detection PCR. The binding positions of the repo overnight fluorescent dye and the quencher fluorescent dye are not particularly limited, but usually, a reporter fluorescent dye is attached to one end (preferably 5, terminal) of the oligonucleotide portion of the probe, and the other end is attached to the other end. Quencher-fluorescent dye is bound. Methods for binding a fluorescent dye to an oligonucleotide are known, for example, Noble et al., Nuc. Acids Res. 12: 3387-3403 (1984) and Iyer et al., J. Am. Chem.
Soc. 112:1253-1254 (1990) に記載されている。 Soc. 112: 1253-1254 (1990).
リアルタイム検出 PC R法自体は公知であり、 そのための装置及びキット も市販されているので、 本発明ではこのような市販の装置及びキットを用い ることもできる。 例えば、 特許第 2825976号に記載の方法に従うか、 P Eバイオシステムズ社製の AB I PR I SM 7700配列決定システ ム ·ユーザ—マニュアルに従って、 実施できる。 Since the real-time detection PCR method itself is known, and devices and kits for the method are also commercially available, such commercially available devices and kits can be used in the present invention. For example, the method can be carried out according to the method described in Japanese Patent No. 2825976, or according to the AB PRISM 7700 Sequencing System User Manual manufactured by PE Biosystems.
8) その他の検出法 8) Other detection methods
本発明エラスチン遺伝子の SNPs またはハプロタイプの検出は、 DNA の塩基配列決定法又は変異の検出法として、 一般に知られている、 以下のよ うな各種方法によっても実施することができる。 Detection of SNPs or haplotypes of the elastin gene of the present invention can also be carried out by the following various methods generally known as DNA base sequence determination methods or mutation detection methods.
(a) 配列特異的オリゴヌクレオチドを用いる PCR— S SO法;各変異に 対するプローブを担体に固相化し、 これに検体 (遺伝子増幅産物）を八イブリ ダイズさせ、 ミスマッチの有無によるハイブリダゼ一ションの効率の差を判 定する方法。 (a) PCR-SSO method using sequence-specific oligonucleotides: A probe for each mutation was immobilized on a carrier, and a sample (gene amplification product) was hybridized to the carrier, and hybridization was determined based on the presence or absence of mismatch. A method to determine the difference in efficiency.
(b) 点変異を検出する P CR— S S P法；点変異に対応する塩基を 3 ' 末端に設定した遺伝子増幅用配列特異的プライマーを用いて、 プライマ一の 3 ' 末端が相補的であるか否かによって PC Rによる増幅効率に著しい差が 生じることを利用する方法。 (b) PCR-SSP method for detecting point mutation; using primers specific to the point mutation at the 3 'end for gene amplification sequence, whether the 3' end of the primer is complementary A method that utilizes the fact that there is a significant difference in amplification efficiency due to PCR depending on whether or not it is not.
(c) PCR— DGGE (変性剤濃度勾配ゲル電気泳動）法；変異 DNA断
片と正常 DNA断片とを混合してハイブリッド結合させた後、 尿素、 ホルム アミドなどの変性剤の濃度が徐々に高くなつているポリアクリルアミドゲル 中で電気泳動すると、 ミスマッチのないホモ 2本鎖に比べて、 より低い濃度 の変性剤の位置で 1本鎖に解離する。 1本鎖 DNAは、 2本鎖 DNAに比べ て泳動速度が速いため、 移動度の差を比較することで 1塩基の変異を検出す ることができる。 (c) PCR—DGGE (denaturing gradient gel electrophoresis) method; After hybridizing the fragment and the normal DNA fragment and performing electrophoresis on a polyacrylamide gel in which the concentration of denaturing agents such as urea and formamide is gradually increasing, a homoduplex without mismatches is obtained. In comparison, dissociation into single strands occurs at lower concentrations of denaturants. Since single-stranded DNA has a higher electrophoresis speed than double-stranded DNA, a single-base mutation can be detected by comparing the difference in mobility.
(d) P CR— DGGE/GCクランプ法（Shefield'V.C.'et al., Proc. Natl. Acad. Sci. USA, 86, 232- 236 (1989)) ；上記 P C R— D G GE法に加え て、 GC含量の高い領域を変異核酸の検出対象である DNA断片につなげる ことにより複数の塩基置換、 欠失、 付加および挿入がある場合の検出の欠点 を補った方法である。 該方法は特に変異検出の対象 DN A断片に GCクラン プを付加する工程を必要とする。 (d) PCR—DGGE / GC clamp method (Shefield'VC'et al., Proc. Natl. Acad. Sci. USA, 86, 232-236 (1989)); This method compensates for the shortcomings of detection when there are multiple base substitutions, deletions, additions and insertions by connecting a region with a high GC content to a DNA fragment from which a mutant nucleic acid is to be detected. This method particularly requires a step of adding a GC clamp to the DNA fragment to be subjected to mutation detection.
(e) P CR— DGGE法に加えて、 T 4エンドヌクレアーゼを用いる核 酸ミスマッチ検出 （米国特許第 6， 183， 958号） (e) Nucleic acid mismatch detection using T4 endonuclease in addition to the PCR-DGGE method (US Patent No. 6,183,958)
T 4エンドヌクレア一ゼの欠失および挿入などの一塩基ミスマッチを含ん でいるへテロ二本鎖 D N Aを認識し、 切断する特性を利用した方法であって、 被検サンプル中の変異を含む DN A断片と標識した配列特異的プローブとの 混合液を P CR増幅した後、 T4エンドヌクレア一ゼによって、 変異部位で DNA断片を切断後、 更に別の配列特異的プローブと切断された DNAとを 反応させた後、 再度 T4エンドヌクレア一ゼによって、 変異部位で DNA断 片を切断し、 各々の切断によって分離され切断産物をポリアクリルアミドゲ ル中で電気泳動し、 生成した泳動パターンを比較することによって、 被検サ ンプル中の 2つの変異が存在するかどうかを検出することができる。 A method that utilizes the property of recognizing and cleaving hetero-double-stranded DNA containing single base mismatches such as deletion and insertion of T4 endonuclease, and which contains DNA in a test sample containing a mutation. After PCR-amplifying the mixture of the fragment and the labeled sequence-specific probe, the DNA fragment is cleaved at the mutation site with T4 endonuclease, and further reacted with another sequence-specific probe and the cleaved DNA. After that, the DNA fragments are again cut at the mutation site by T4 endonuclease, and the cleavage products separated by each cleavage are electrophoresed in polyacrylamide gel, and the generated electrophoretic patterns are compared to obtain a target. It can detect whether two mutations are present in the test sample.
該方法によれば、 多型遺伝子のハプロタイプを決定できるだけでなく、 D N Aサンプルから、 父方のハプロタイプと母方のハプロタイプを特定するこ とも可能である。 According to the method, not only can the haplotype of the polymorphic gene be determined, but also the paternal haplotype and the maternal haplotype can be specified from the DNA sample.
( f ) RN a s e保護アツセィ法（Finkelsiein, J.， ei al. , Genomics, 7, 167-172(1990)) (f) RNase protection atssey law (Finkelsiein, J., ei al., Genomics, 7, 167-172 (1990))
(g) in situ RT-PCR(Nucl. Acids Res., 21， 3159-3166 (1993))
(h) サザンブロッテイング（Sambrook, J., et al., Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press :NY. (1989))(g) In situ RT-PCR (Nucl. Acids Res., 21, 3159-3166 (1993)) (h) Southern blotting (Sambrook, J., et al., Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press: NY. (1989))
( i)ドットハイブリダィゼ一シヨン法 （Southern, E. M. ， J. Mol. Biol., 98:503-517 (1975)など参照) 、 (i) Dot hybridization method (see Southern, EM, J. Mol. Biol., 98: 503-517 (1975), etc.),
( j ) 蛍光 i n s i t u ハイブリダィゼーシヨン（FISH:Takahashi Ε· , et al., Hum. Genet., 86, 1416 (1990)) (j) Fluorescence insitu hybridization (FISH: Takahashi Ε, et al., Hum. Genet., 86, 1416 (1990))
(k) 競合的ゲノミック ·ハイブリダィゼ一シヨン（Co即 arative Genomic Hybridization:CGH: Kallioneimi, A. , et al. , Science, 258, 818- 821 (1992))、 (Spectral karyotyping: SKY: Rowley, J.D. , et al., Blood, 93, 2038-2042 (1999)) (k) Competitive genomic hybridization (Co-immediate arative Genomic Hybridization: CGH: Kallioneimi, A., et al., Science, 258, 818-821 (1992)), (Spectral karyotyping: SKY: Rowley, JD, et al., Blood, 93, 2038-2042 (1999))
(1) 酵母人工染色体 （YAC) ベクタ一のクローンをプロ一ブとする方法 (Lengauer, C. , et al., Cancer Res., 52, 2590—2596(1992))。 (1) A method of using a yeast artificial chromosome (YAC) vector clone as a probe (Lengauer, C., et al., Cancer Res., 52, 2590-2596 (1992)).
(m) NA S B A法 [Nucleic acid sequence-based amplification, Nature, 350, 91-92 (1991)〕 。 (m) NASB A method [Nucleic acid sequence-based amplification, Nature, 350, 91-92 (1991)].
これらの中で、 好ましい方法としては、 一塩基プライマー伸長法、 具体的 にはスナップショット法またはピロシーケンス法を挙げることができる。 Among them, preferred methods include a single-base primer extension method, specifically, a snapshot method or a pyrosequencing method.
ここで上記遺伝子検出法に用いられるプライマーとしては、 本発明遺伝子 ハプロタイプを含む領域のみを特異的に増幅できるものである限り、 特に制 限はなく、 本発明遺伝子の配列情報に基づいて適宜設計することができる。 好適には、 例えば、 15〜 50ヌクレオチド程度の長さを有し、 本発明遺伝 子の各八プロタイプ配列の S N Pを挟むように合成されたヌクレオチド配列 を有するものなどが挙げられる。 或いは、 一塩基多型部位よりもセンス鎖又 はアンチセンス鎖において 1〜数塩基上流 （5' 側） に 3' 末端を有する上 記プライマーなどが挙げられる。 Here, the primer used in the gene detection method is not particularly limited as long as it can specifically amplify only the region containing the haplotype of the gene of the present invention, and is appropriately designed based on the sequence information of the gene of the present invention. be able to. Preferably, for example, those having a length of about 15 to 50 nucleotides and having a nucleotide sequence synthesized so as to sandwich the SNP of each of the eight prototypic sequences of the gene of the present invention are exemplified. Alternatively, the above-mentioned primers having a 3 ′ end one to several bases upstream (5 ′ side) in the sense strand or the antisense strand from the single nucleotide polymorphism site may be used.
本発明においては、 イントロン 20の遺伝子多型部位とイントロン 23の 遺伝子多型部位を同時に同定できるように、 上記検出方法を適宜設定しても よい。 また、 各々の遺伝子多型を別々に同定できるように設定してもよい。 本発明の遺伝子ハプロタイプには、 本発明にかかる多型遺伝子を検出する ための特異的プライマー及びノ又は特異的プローブとして使用される D N A
断片もまた包含される。 In the present invention, the above detection method may be appropriately set so that the gene polymorphism site of intron 20 and the gene polymorphism site of intron 23 can be simultaneously identified. In addition, it may be set so that each gene polymorphism can be identified separately. The gene haplotype of the present invention includes a specific primer and a DNA used as a specific probe for detecting the polymorphic gene according to the present invention. Fragments are also included.
また、 本発明においては、 所望の遺伝子における脳動脈瘤の発症リスクの 存在を検出できることから、 例えば、 脳動脈瘤発症リスクの存在の診断にお いて、 検出用遺伝子特異的プロ一ブとして、 エラスチン遺伝子のイントロン 2 0の該遺伝子多型部位が野生型であって、 イントロン 2 3の該遺伝子多型 部位が変異型である八プロタイプを検出するような多型特異的配列プローブ を作成することなども挙げられる。 Further, in the present invention, since the presence of a risk of developing a cerebral aneurysm in a desired gene can be detected, for example, in the diagnosis of the presence of a risk of developing a cerebral aneurysm, elastin is used as a gene-specific probe for detection. To prepare a polymorphism-specific sequence probe for detecting an octatype in which the polymorphic site of intron 20 of the gene is wild-type and the polymorphic site of intron 23 is a mutant type. And the like.
具体的には、 エラスチン遺伝子のイントロン 2 0の 1 7番目の核酸配列が 野生型か変異型であるかどうか、 即ち、 1 7番目の核酸が Tであるか Cであ るかを検出できる特異プローブ、 イントロン 2 3部位の 2 4番目の核酸配列 が野生型か変異型であるかどうか、 即ち、 2 4番目の核酸が Tであるか で あるかを検出できる特異的プロ一ブなどが挙げられる。 キッ卜 Specifically, a 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, that is, whether the 17th nucleic acid is T or C Probes, specific probes that can detect whether the 24th nucleic acid sequence at intron 23 site is wild-type or mutant, that is, whether the 24th nucleic acid is T or not. Can be Kit
本発明の同定方法は、 試料中のヒト ·エラスチン遺伝子のイントロン 2 0 およびイントロン 2 3のハプロタイプ検出のための試薬キットを利用するこ とによって、 簡便に実施することができる。 本発明は、 かかるキットも提供 するものである。 具体的には、 本発明は、 ヒト ·エラスチン遺伝子の 2 0番 目のイントロン部位 D N A配列の 1 7番目の D N Aの遺伝子多型検出用ブラ イマ一、 及びヒト ·エラスチン遺伝子の 2 3番目のイントロン部位 D NA配 列の 2 4番目の D N Aの遺伝子多型検出用プライマ一、 を含む脳動脈瘤の発 症のリスク検出用診断キットを提供するものである。 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 in the 17th DNA of the DNA sequence of the 20th intron of the human elastin gene, and a 23rd intron of the human elastin gene. It is intended to provide a diagnostic kit for detecting the risk of developing a cerebral aneurysm, comprising a primer for detecting a gene polymorphism of the 24th DNA of the site DNA sequence.
プライマーとしては、 上記エラスチン遺伝子のセンス鎖又はアンチセンス 鎖にハイプリダイズすることができ、 且つィントロン 2 0およびィントロン 2 3の各遺伝子多型部位を増幅することが可能な各プライマー、 遺伝子多型 部位の 1塩基から数塩基上流 （5 ' 側） に 3 ' 末端を有する配列からなるプ ライマー、 遺伝子多型部位を有する配列からなるプライマーなどが例示でき る。 As the primers, each primer capable of hybridizing to the sense strand or antisense strand of the elastin gene and capable of amplifying each of the gene polymorphism sites of intron 20 and intron 23, a gene polymorphism site Examples of such primers include a primer having a sequence having a 3 ′ end one to several bases upstream (5 ′ side) from the base and a primer having a sequence having a gene polymorphism site.
更に、 遺伝子多型部位を含む数塩基の核酸配列を認識する制限酵素を含有
するヒト脳動脈瘤発症のリスク検出の検出用試薬キットが提供される。 In addition, contains a restriction enzyme that recognizes a nucleic acid sequence of several bases including the polymorphic site The present invention provides a reagent kit for detecting the risk of developing a human cerebral aneurysm.
該試薬キットは、 少なくともエラスチン遺伝子のイントロン 2 0部位およ びイントロン 2 3部位の D N A塩基配列もしくはその相補的塩基配列の一部 または全てに八イブリダィズする D NA断片を、 必須構成成分として含んで いれば、 他の成分として、 標識剤、 P C R法に必須な試薬 （例えば、 T a q The reagent kit contains, as an essential component, a DNA fragment that at least partially hybridizes to a part or all of the DNA base sequence of at least intron 20 and intron 23 of the elastin gene or its complementary base sequence. If present, labeling agents and other reagents essential for PCR (eg, Taq
D N Aポリメラーゼ、 デォキシヌクレオチド三リン酸、 プライマーなど） が 含まれていてもよい。 DNA polymerase, deoxynucleotide triphosphates, primers, etc.).
標識剤としては、 放射性同位元素または蛍光物質などの化学修飾物質など が挙げられるが、 D N A断片自身が予め該標識剤でコンジュゲートされてい てもよい。 更に、 当該試薬キットには、 測定を実施する際の便益のために適 当な反応希釈液、 標準抗体、 緩衝液、 洗浄剤、 反応停止液などが含まれてい てもよい。 Examples of the labeling agent include a chemical modifying substance such as a radioisotope or a fluorescent substance, and the DNA fragment itself may be conjugated with the labeling agent in advance. Furthermore, the reagent kit may include a reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., which are appropriate for the benefit of performing the measurement.
更に本発明は、 前記測定方法を用いるヒト脳動脈瘤発症のリスク診断方法 および該方法に用いる診断剤並びに診断用キットをも提供するものである。 本発明の遺伝子治療 Further, the present invention also provides a method for diagnosing the risk of developing human cerebral aneurysm using the above-mentioned measurement method, a diagnostic agent used for the method, and a diagnostic kit. Gene therapy of the present invention
また、 本発明は、 ヒト ·エラスチン遺伝子のイントロン 2 3の 2 4番目の 核酸配列を野生型に変換するためのオリゴ D N A、 あるいは該オリゴ D N A を含有する遺伝子治療用ベクターおよび該ベクタ一によりヒト ·エラスチン 遺伝子のイントロン 2 3の 2 4番目の核酸配列を野生型に変換するためのォ リゴ D N Aを導入した細胞を有効成分とする医薬を提供しょうとするもので ある。 Further, the present invention provides an oligo DNA for converting the 24th nucleic acid sequence of intron 23 of the human elastin gene into a wild type, or a gene therapy vector containing the oligo DNA and a human An object of the present invention is to provide a drug containing, as an active ingredient, cells into which oligo DNA for converting the nucleic acid sequence at position 24 of intron 23 of the elastin gene to wild type has been introduced.
即ち、 本発明によれば、 数十から数百塩基からなり、 ヒト ·エラスチン遺 伝子の部分配列からなり、 イントロン 2 3の 2 4番目の核酸配列を Tである ように合成されたォリゴヌクレオチド、 該ォリゴヌクレオチドを含有する遺 伝子治療用導入用ベクターおよび該ベクターにより該オリゴヌクレオチドを 導入した細胞、 並びに該遺伝子治療用導入用ベクター又は該ベクタ一により ヒト ·エラスチン遺伝子のイントロン 2 3の野生型核酸を導入した細胞を有 効成分とする遺伝子治療剤などが提供される。
また、 上記オリゴヌクレオチド、 該オリゴヌクレオチドを含有する遺伝子 治療用ベクター、 該オリゴヌクレオチド又はべクタ一を導入した細胞、 並び に該遺伝子治療用導入用ベクターまたは該ベクターによりヒト ·エラスチン 遺伝子のィントロン 23の野生型核酸を導入した細胞を有効成分とする遺伝 子治療剤を、 エラスチン遺伝子のイントロン 20の 17番目の核酸が野生型 であって、 イントロン 23の 24番目の核酸が Cに変異している変異型であ るハプロタイプを有する脳動脈瘤のリスクのある人、 あるいは該ハプロタイ プを有する脳動脈瘤（クモ膜下出血)患者の脳血管細胞または患者の末梢血組 織部位に、 投与することによって、 これら組織における遺伝子変異を改善し、 脳動脈瘤発症の二次予防するか、 あるいは脳動脈瘤（クモ膜下出血)患者の再 発を抑制することを特徴とする脳動脈瘤予防および治療剤を提供することが できる。 That is, according to the present invention, an oligo comprising several tens to several hundreds of bases, comprising a partial sequence of the human elastin gene, and being synthesized so that the 24th nucleic acid sequence of intron 23 is T Nucleotides, transfer vectors for gene therapy containing the oligonucleotides, cells transfected with the oligonucleotides with the vectors, and introns of the human elastin gene with the transfer vectors for gene therapy or the vector. And a gene therapy agent comprising a cell into which the wild-type nucleic acid is introduced as an active ingredient. The oligonucleotide, the gene therapy vector containing the oligonucleotide, the cell into which the oligonucleotide or the vector is introduced, and the human elastin gene intron 23 by the gene therapy introduction vector or the vector. A gene therapy drug containing a cell into which a wild-type nucleic acid has been introduced as an active ingredient is a mutation in which the 17th nucleic acid of intron 20 of the elastin gene is wild-type and the 24th nucleic acid of intron 23 is mutated to C. At risk for a cerebral aneurysm having a haplotype, or a patient with a cerebral aneurysm (subarachnoid hemorrhage) having the haplotype, or to a peripheral blood tissue site of the patient. Improve gene mutations in these tissues to prevent secondary development of cerebral aneurysms or It is possible to provide a prophylactic and therapeutic agent for cerebral aneurysms characterized by suppressing recurrence in patients with subarachnoid hemorrhage.
本発明の実施には、 特記しないかぎり、 化学、 分子生物学、 微生物学、 組 換え DNA、 遺伝学及び免疫学の慣用的な方法を用いることができる。 例え ぱ、 マニアテイス (Maniat is, T.， et al. , Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, cold Spring Harbor, New York (1982)) 、 サムブルッ ク （Sambrook, J.， et al. , Molecular Cloning a Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1981))、 ァウスベル（ausbel'F.M.， et al. , Current Protocols in Molecular Biology, John Wiley and Sons, New York, New York, (1992)) , グロ一バ一 （Glover, D.， D N A cloning, I and II (Oxford Press) (1985))、 アナンド (Anand, Techniques for the analysis of com lex genomes, (Academic Press (1992)) , グスリ一 (Guthrie, g. , et al. , Guide to Yeast Genetics and Molecular Biology, (Academic Press) (1991))及びフィ ンク （Fink，et al., Hum. Gene Ther., 3, 11- 19 (1992))などの方法を用いることができる。 The practice of the present invention can employ, unless otherwise indicated, conventional methods of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. For example, Mania is, T., et al., Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, cold Spring Harbor, New York (1982)), Sambrook, J., et al., Molecular Cloning a Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1981)), Ausbel (ausbel'FM, et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, New York, (1992)), Glover, D., DNA cloning, I and II (Oxford Press) (1985)), Anand, Anand, Techniques for the analysis of com lex genomes, (Academic Press ( 1992)), Guthrie, g., Et al., Guide to Yeast Genetics and Molecular Biology, (Academic Press) (1991)) and Fink (Fink, et al., Hum. Gene Ther., 3, 11-19 (1992)).
本発明エラスチン遺伝子八プロタイプ検出のための遺伝子解析は、 上述の 判定方法などによって行なうことができ、 例えば、 エラスチン遺伝子のイン トロン 20部位が野生型であって、 イントロン 23が変異型であるハプロ夕
イブを検出するために、 ヒトの血液または組織細胞からの細胞から得られた ゲノム D N Aをエラスチン遺伝子のイントロン 2 0の 1 7番目とイントロン 2 3の 2 4番目の核酸配列のそれぞれを含むように、 プライマ一を作成し、 D N Aを増幅し、 スクリ一ニングすることによって、 ヒト ·エラスチン遺伝 子八プロタイプの有無を確認することができる。 Gene analysis for detecting the eight elastin gene prototypes of the present invention can be performed by the above-described determination method and the like. For example, a haplotype in which intron 20 of the elastin gene is a wild type and intron 23 is a mutant type evening To detect Eve, genomic DNA obtained from cells from human blood or tissue cells was modified to include the elastin gene intron 20 17 and intron 23 24 nucleic acid sequences, respectively. By preparing a primer, amplifying DNA, and screening, the presence of the eight human elastin gene prototype can be confirmed.
これらの診断は、 ヒトのいかなる組織から得た D N Aを用いても調べるこ とが出来る。 好適な方法としては、 血液を取り、 血液細胞から D NAを抽出 する方法、 標的とする組織細胞から直接得られた細胞から D N Aを抽出する 方法などが挙げられる。 These diagnoses can be examined using DNA obtained from any human tissue. Suitable methods include a method of extracting blood and extracting DNA from blood cells, and a method of extracting DNA from cells obtained directly from target tissue cells.
イントロン 2 0の該遺伝子多型部位が野生型でィントロン 2 3の該遺伝子 多型部位が変異型であるハプロタイプを有していたヒトに対しては、 例えば、 次のような遺伝子治療を行なうことができる。 イントロン 2 3の遺伝子多型 部位を野生型に変換することができる発現制御エレメントに連結したヒ卜 · エラスチン遺伝子のィントロン 2 3の遺伝子多型部位が野生型である D N A のコピーを含み、 かつ該細胞内で複製出来るウィルスまたはプラスミドべク 夕一を作成する。 ここで適当なベクターは、 米国特許第 5， 2 5 2 , 4 7 9 号及ぴ P C T国際公開 WO 9 3 / 0 7 2 8 2号に開示されたベクターを使用 して作成できる。 次いで作成されたヒト ·エラスチン遺伝子のイントロン 2 3の遺伝子部位が野生型である D N Aを含むべク夕一を血液などの細胞内に 全身的に対象となる患者に注射投与する。 形質転換された遺伝子が標的化血 管細胞の染色体に恒久的に取り込まれない場合には、 該処理を定期的に繰り 返すことができる。 For a human having a haplotype in which the polymorphic site of intron 20 is wild-type and the polymorphic site of intron 23 is mutant, for example, the following gene therapy is performed. Can be. The intron 23 gene polymorphic site of the human elastin gene linked to an expression control element capable of converting the gene polymorphic site of intron 23 to wild-type; Create a virus or plasmid vector that can replicate in cells. Suitable vectors here can be made using the vectors disclosed in US Pat. Nos. 5,252,479 and PCT International Publication WO 93/07282. Next, the prepared vector containing DNA in which the intron 23 gene region of the human elastin gene contains wild-type DNA is injected into cells such as blood, and administered to the target patient systemically. If the transformed gene is not permanently integrated into the chromosome of the targeted vascular cell, the process can be repeated periodically.
遺伝子治療方法は、 前記の如く遺伝子導入用の材料を直接体内に投与する in vivo 法と、 患者の体内より標的とする細胞を一旦取り出して体外で遺伝 子を導入して、 その後細胞を体内に戻す ex vivo 法のどちらの方法も適宜選 択して用いることができる。 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, the gene is introduced outside the body, and then the cell is introduced into the body Any of the ex vivo methods of returning can be appropriately selected and used.
本発明の遺伝子を含有するべクタ一の製造法において、 導入されるエラス チン遺伝子のイントロン 2 3が野生型である D N Aは、 その遺伝子の塩基配 列情報に基づいて、 一般的な遺伝子工学的手法により容易に製造 ·取得する
ことができる olecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989) ；続生化学実験講座 「遺伝子研究法 I、 II、 III」 、 日本生化学会編 (1986) 等参照〕 。 DN Aの合成についても上述した方法を適用できる。 In the method for producing a 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 obtained by general genetic engineering based on the base sequence information of the gene. Easily manufacture and acquire by method Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Seismic Chemistry Laboratory Lecture “Gene Research Methods I, II, III”, edited by The Biochemical Society of Japan (1986), etc.]. The method described above can be applied to the synthesis of DNA.
本発明によれば、 血液または血清のごとき生物学的試料を調製し、 所望に より核酸を抽出し、 ヒト ·エラスチン遺伝子のイントロン 20およびイント ロン 23の八プロタイプ感受性遺伝子が存在するか否かについて分析するこ とも可能である。 According to the present invention, a biological sample such as blood or serum is prepared, nucleic acids are extracted as desired, and whether or not the octaprototype-sensitive genes of human elastin gene intron 20 and intron 23 are present. It is also possible to analyze
また、 本発明によれば、 血液または血清のごとき検体から得られるエラス チン遺伝子のイントロン 20およびイントロン 23のハプロタイプを分析す ることにより、 脳動脈瘤発症のリスクの存在を検出することができる。 図面の簡単な説明 Further, according to the present invention, the presence of a risk of cerebral aneurysm development can be detected by analyzing the haplotypes of intron 20 and intron 23 of the elastin gene obtained from a sample such as blood or serum. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 染色体 5番における多点連鎖解析によるマ一力一と最大 1 odを 示す図面である。 FIG. 1 is a drawing showing the maximum power and maximum 1 od by multipoint linkage analysis on chromosome 5.
図 2は、 染色体 7番における多点連鎖解析によるマーカ一と最大 1 o dを 示す図面である。 FIG. 2 is a drawing showing one marker and a maximum of 1 od by a multipoint linkage analysis on chromosome 7.
図 3は、 染色体 14番における多点連鎖解析によるマーカーと最大 1 o d を示す図面である。 FIG. 3 is a drawing showing a marker of chromosome 14 by multipoint linkage analysis and a maximum of 1 od.
図 4は、 エラスチン遺伝子 （ELN) の構造を示す図面である。 PMはプ 口モーター、 EXはェキソン、 I NTはイントロンを示す。 FIG. 4 is a drawing showing the structure of the elastin gene (ELN). PM stands for motor, EX stands for exon, and INT stands for intron.
図 5は、 ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DN A配列 における 1 7番目の DN Aの遺伝子多型 （野生型： T、 変異型： C) を示す 図面である。 FIG. 5 is a drawing showing the 17th DNA polymorphism (wild type: T, mutant type: C) in the DNA sequence at the 20th intron site of the human elastin gene.
図 6は、 ヒト ·エラスチン遺伝子の 23番目のイントロン部位 DNA配列 における 24番目の DNAの遺伝子多型 （野生型： Τ、 変異型： C) を示す 図面である。
発明を実施するための最良の形態 FIG. 6 is a drawing showing the polymorphism of the 24th DNA in the DNA sequence of the 23rd intron of the human elastin gene (wild type: Τ, mutant type: C). BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を更に詳しく説明するため、 実施例を挙げるが、 本発明はこ れに限定されない。 実施例 1 ゲノム全域での連鎖解析 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
材料および方法： Materials and methods:
全国 1 1 0 0ケ所の脳外科関連施設の協力により、 1 0 4対の罹患同胞対 (脳動脈瘤患者同胞）を集めた。 その内訳は、 8 5家系収集され、 7 7組の 2 人兄弟、 7組の 3人兄弟、 1組の 4人兄弟であった。 また、 7 3家系で発端 者がクモ膜下出血であり、 1 2家系で発端者が未破裂脳動脈瘤のみを認めた (Kasuya, H.， et al .， Neurosurgery, 46 (6) 1301-1306 (2000) ) 0 遺伝要因 の強さの指標である、 兄弟での相対危険率 （A s= 6 ) を考慮したシュミレー ションにより、 この同胞対数で十分遺子座を特定できると予想されたので、 罹患同胞対連鎖解析法を実施した。 With the cooperation of 110 neurosurgery-related facilities nationwide, 104 pairs of affected sibs (branchial aneurysm patients) were collected. The breakdown consisted of 85 families, 77 pairs of 2 brothers, 7 pairs of 3 brothers, and 1 pair of 4 brothers. The proband had subarachnoid hemorrhage in 73 families, and the proband only had unruptured cerebral aneurysms in 12 families (Kasuya, H., et al., Neurosurgery, 46 (6) 1301- 1306 (2000)) 0 Simulations considering the relative risk factor (A s = 6) of siblings, which is an indicator of the strength of genetic factors, were expected to be enough to identify the orbital locus using this log of siblings. Therefore, an affected sib-pair linkage analysis was performed.
即ち、 該方法の原理を略記すると、 罹患している兄弟は病気の原因となる ァレルを親から受け継いでいるため、 そのアレルを必ず共有することとなる 一方、 兄弟が共有するアレルの数は 1である（帰無仮説に基づく値）。 多くの 罹患同胞対で共有するアレル数を検討することにより、 帰無仮説に基づくァ レル数より、 多くのアレル共有が観察できたとき、 「連鎖を認めた」 という こととなる。 Briefly, the principle of the method is as follows. Since the affected sibling inherits the allele that causes the disease from the parent, the allele must be shared, while the number of alleles shared by the sibling 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 has been recognized".
全ゲノムを網羅する連鎖解析の目的で 4 0 0個の蛍光ラベルされたマイク ロサテライ トマ一力一からなるリンケ一ジマッピングセッ ト（Linkage mapping set vers ion I I. P Eアプライド ·バイオシステムズ社製）を用い た。 このマーカーセットによれば、 平均 8 . 6センチモルガンの間隔で、 解 析を行なうことができる。 しかしながら、 日本人においてはへテロ接合性の 低いマーカ一が存在していたので、 さらにマーカーを増やし、 4 2 0マーカ 一を用いてゲノム全域での解析を実施した。 Linkage mapping set version I I. PE Applied Biosystems, Inc., consisting of 400 fluorescently labeled microsatellites for the purpose of linkage analysis covering the entire genome Was used. According to this marker set, analysis can be performed at an average interval of 8.6 centiMorgan. However, since there was a marker with low heterozygosity in Japanese, the number of markers was further increased, and analysis of the entire genome was performed using 420 markers.
3種類の 6— F AM (青色）、 H E X (緑色）、 N E D (黄色）の蛍光色素で標 識されたプライマ一の使用により、 多型性に富む 2塩基繰り返し配列（ジヌク
レオチドリピート ·マイクロサテライト）を PCR増幅することができた。 かくして得られた PC R産物のサイズの違いを自動解析装置 A B I PR I SM : 377モデル（アプライド ·バイオシステムズ社製）を用いたシ一ケ ンスゲルで確認した。 前記 3種類の蛍光色素で標識したプライマーを用いて P CR反応を行なっているので、 たとえ同一サイズの P CR産物であっても 3種類の色を別々に判別できる。 前記反応ゲルにて 96サンプルに対して、 15種類のマ一カーについて同時に解析可能であり、 約 1500個の遺伝子 タイピングを 1回の電気泳動で処理することができた。 By using primers labeled with three types of 6-FAM (blue), HEX (green), and NED (yellow) fluorescent dyes, a highly dimorphic dinucleotide repeat (dinuc) Leotide repeat microsatellite) could be amplified by PCR. The difference in the size of the PCR product thus obtained was confirmed on a sequence gel using an automatic analyzer ABI PRISM: Model 377 (manufactured by Applied Biosystems). Since the PCR reaction is performed using the primers labeled with the above three kinds of fluorescent dyes, even if the PCR products have the same size, the three kinds of colors can be distinguished separately. With the above-mentioned reaction gel, 96 kinds of samples could be analyzed simultaneously for 15 kinds of markers, and about 1500 genotyping could be processed by one electrophoresis.
大量のサンプルのタイピングは、 Ge n e S c an、 Ge n oTy e r プログラム（PEアプライド社製）を用いて迅速処理を行なった。 Typing of a large amount of samples was performed rapidly using the GeneScan and the GenoTyer program (manufactured by PE Applied).
連鎖の検定は、 ノンパラメトリック解析である GENEHUNTER (KruglyaK, L. ei al. Am. J. Genet., 58, 1347-1363 (1996)), および S I B P A Lプログラム（S I B P A L : SAGE, Statistical Analysis for Genetic Epidemiology, release 3.1. Department of Biometry and Genetics. LSU Medical Center , New Orleans, LA)を用いて行なった。 解析結果： The linkage test is performed using the nonparametric analysis GENEHUNTER (KruglyaK, L. ei al. Am. J. Genet., 58, 1347-1363 (1996)), and the SIBPAL program (SIBPAL: SAGE, Statistical Analysis for Genetic Epidemiology, release 3.1. Department of Biometry and Genetics. LSU Medical Center, New Orleans, LA). Analysis result:
連鎖が認められる領域の判定は、 以下に示すランダー（Lander)およびクラ ッダリヤック（Kruglyak) のガイドライン（Nature Genet, 11， 241 (1995))に従 つて、 偽陽性の連鎖を得る基準を基にした。 さらにゲノム全域の連鎖解析で は偽陽性が多いため、 有意差 P<0. 05のマ一力一が 2つ以上連続して認 められた領域を連鎖ありと判定した。 ランダ一およびクラッダリャックのガイドライン： The determination of regions with linkage is based on the criteria for obtaining false-positive linkages according to the following Lander and Kruglyak guidelines (Nature Genet, 11, 241 (1995)). . Furthermore, in the linkage analysis of the entire genome, since there are many false positives, a region in which two or more successively significant differences with a significant difference P <0.05 are recognized as having linkage was found. Guidelines for Landa and Claddaryak:
多因子疾患でゲノム全域での連鎖解析が盛んにおこなわれるようになって きたが、 個々の遺伝子の連鎖解析については、 その遺伝子機能が原因となり うるかといつた判断も必要となる。 しかしながらゲノム全域での解析では遺 伝子機能はその段階で考慮に入れられないので、 純粋に数理遺伝学的な有意 の判断基準（閾値）が求められる。 そこで彼らはシユミレ一シヨンに従い、 有
意な連鎖基準を設けている 。 Linkage analysis across the genome has been actively performed in multifactorial diseases, but linkage analysis of individual genes requires constant judgment as to whether the gene function may be the cause. However, in gene-wide analysis, gene function is not taken into account at that stage, so a purely mathematical and genetic significance criterion (threshold) is required. So they follow the rules, We have established a chain standard.
- Suggestive linkage: P<7. 4 x 10— 4、 1 o d>2. 2、 ゲノム全域で 1個の偽陽性の連鎖結果を得る基準 - Suggestive linkage:. P <. 7 4 x 10- 4, 1 o d> 2 2, reference to obtain a chain result in one false positive in genome-wide
- Significant linkage: P<2. 2 x 10— 5、 1 o d> 3. 6、 ゲノム全域で 0. 05個の偽陽性の連鎖結果を得る基準 - Significant linkage: P <. 2 2 x 10- 5, 1 o d> 3. 6, a reference to obtain a chain result of 0.05 pieces of false positives in genome-wide
- High Significant linkage: Pく 3. 0 x 10一7、 1 o d> 5. 4、 ゲノム全域で 0. 01個の偽陽性の連鎖結果を得る基準 - High Significant linkage: P rather than 3. 0 x 10 one 7, 1 o d> 5. 4 , the reference to obtain a chain result of 0.01 pieces of false positives in genome-wide
全染色体スクリーニングの結果 （ここで、 S I BPALでの解析では X染 色体は含まない。 ) から、 5番 (D5S428,D5S644)、 7番 (D7S669, D7S630)、 1 4番染色体（D14S258，D14S74)で連鎖を めた。 5番 （Chromosome 5) 、 7番 From the results of the whole chromosome screening (X chromosome is not included in the analysis using SI BPAL), chromosomes 5 (D5S428, D5S644), 7 (D7S669, D7S630), and 14 chromosomes (D14S258, D14S74) ). No. 5 (Chromosome 5), No. 7
(Chromosome 7) 及び 14番 （Chromosome 14) 染色体での結果を表 1に示す。
(Chromosome 7) and No. 14 (Chromosome 14) chromosomes are shown in Table 1.
表 1 table 1
Chromosome 5 Chromosome 5
Marker cM HZ Pairs 励 t -values Marker cM HZ Pairs encouraging t -values
D5S1981 0.6 0.76 83 0.51 0.47 0.321D5S1981 0.6 0.76 83 0.51 0.47 0.321
D5S406 10.7 0.73 82 0.51 0,43 0.333D5S406 10.7 0.73 82 0.51 0,43 0.333
D5S630 18.6 0.90 83 0.52 0.46 0.322D5S630 18.6 0.90 83 0.52 0.46 0.322
D5S416 27.9 0.64 81 0.50 0.07 0.473D5S416 27.9 0.64 81 0.50 0.07 0.473
D5S419 39.5 0.87 80 0.47 -1.02 0.846D5S419 39.5 0.87 80 0.47 -1.02 0.846
D5S426 51.6 0.78 82 0.48 -0.75 0.772D5S426 51.6 0.78 82 0.48 -0.75 0.772
D5S418 58.1 0.78 81 0.54 1.19 0.12D5S418 58.1 0.78 81 0.54 1.19 0.12
D5S407 65 0.86 83 0.52 0.48 0.316D5S407 65 0.86 83 0.52 0.48 0.316
D5S647 74.7 0.82 83 0.54 1.25 0.108D5S647 74.7 0.82 83 0.54 1.25 0.108
D5S424 82.8 0.68 82 0.52 0.86 0.196D5S424 82.8 0.68 82 0.52 0.86 0.196
D5S641 92.3 0.81 82 0.51 0.33 0.371D5S641 92.3 0.81 82 0.51 0.33 0.371
D5S428 95.4 0.68 81 0.57 3.09 o.oo r*D5S428 95.4 0.68 81 0.57 3.09 o.oo r *
D5S644 104.5 0.83 81 0.56 2.14 0.018*D5S644 104.5 0.83 81 0.56 2.14 0.018 *
D5S433 112.2 0.75 83 0.53 1.08 0.141D5S433 112.2 0.75 83 0.53 1.08 0.141
D5S2027 118.9 0.59 82 0.52 0.79 0.216D5S2027 118.9 0.59 82 0.52 0.79 0.216
D5S471 129.6 0.71 83 0.53 1.40 0.083 D5S471 129.6 0.71 83 0.53 1.40 0.083
Chromosome 7 Chromosome 7
Marker cM HZ Pairs IBD t -values Marker cM HZ Pairs IBD t -values
D7S531 4.8 0.77 81 0.53 1.14 0.130D7S531 4.8 0.77 81 0.53 1.14 0.130
D7S517 7.8 0.79 83 0.54 1.55 0.063D7S517 7.8 0.79 83 0.54 1.55 0.063
D7S513 17.7 0.9 83 0.53 0.89 0.189D7S513 17.7 0.9 83 0.53 0.89 0.189
D7S507 29.1 0.82 83 0.50 -0.09 0.538D7S507 29.1 0.82 83 0.50 -0.09 0.538
D7S493 35 0.73 83 0.49 -0.29 0.615D7S493 35 0.73 83 0.49 -0.29 0.615
D7S516 42.1 0.76 83 0.49 -0.24 0.596D7S516 42.1 0.76 83 0.49 -0.24 0.596
D7S484 55.6 0.79 82 0.51 0.34 0.367D7S484 55.6 0.79 82 0.51 0.34 0.367
D7S510 60.5 0.82 83 0.55 1.87 0.032*D7S510 60.5 0.82 83 0.55 1.87 0.032 *
D7S519 70.5 0.74 83 0.53 1.32 0.095D7S519 70.5 0.74 83 0.53 1.32 0.095
D7S502 79.6 0.85 82 0.54 1.13 0.131D7S502 79.6 0.85 82 0.54 1.13 0.131
D7S669 90.9 0.83 83 0.56 2.06 0.021*D7S669 90.9 0.83 83 0.56 2.06 0.021 *
D7S630 98.7 0.77 82 0.55 1.77 0.040*D7S630 98.7 0.77 82 0.55 1.77 0.040 *
D7S657 105.2 0.77 83 0.54 1.52 0.066
D7S657 105.2 0.77 83 0.54 1.52 0.066
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続いて、 連鎖を認めた上記 3箇所の領域について、 さらにマーカーを増や し、 連鎖領域を狭めることと、 確実にするための GENEHUTERによる 多点連鎖解析をおこなった。 6C9990 / Z0 OAV Subsequently, for the three regions where linkage was recognized, markers were further increased to reduce the linkage region, and multipoint linkage analysis using GENEHUTER was performed to ensure the linkage region.
その結果を図 1から図 3に示す。 その結果、 図 1に示されるように染色体 5番で最大 l o d 2. 24、 図 2に示されるように染色体 7番で最大 1 o d 3. 22、 図 3に示されるように染色体 14番で最大 1 o d 2. 31を得た。 詳細なマツビングの結果、 7番染色体のマーカー D 7 S 2472において、 P=0. 000027と最も強い連鎖結果を得た。 The results are shown in Figs. As a result, as shown in Figure 1, chromosome 5 had a maximum lod of 2.24, chromosome 7 had a maximum of 1 od 3.22 as shown in Figure 2, and chromosome 14 had a maximum of od 3.22 as shown in Figure 3. 1 od 2.31 was obtained. As a result of the detailed mating, the strongest linkage result was obtained with P = 0.000027 for the marker D7S2472 on chromosome 7.
かくして得られた値は、 上記ランダーおよびクラッグリャックらの Suggestive linkage を満たすものであり、 脳動脈瘤遺伝子座のひとつを染色 体 7 Q 1 1. 23に特定できた。 この結果は、 この領域に疾患遺伝子が存在 することを強く示唆するものであった。 実施例 2 脳動脈瘤候補遺伝子の探索 The value thus obtained satisfies the above-mentioned Suggestive linkage of Lander and Cragglyak, and one of the cerebral aneurysm loci was identified as chromosome 7Q11.23. This result strongly suggested the presence of a disease gene in this region. Example 2 Search for cerebral aneurysm candidate genes
上記実施例 1で得られた最も強い連鎖を認めた 7 q 1 1. 23領域の遺伝 マ一カー、 D 7 S 2472及ぴ D 7 S 2415に隣接している候補遺伝子に つ いて検討 し た結果、 エラ ス チ ン遺伝子が存在 していた (tit tp://www. ncbi. nlm. nih. gov/genemap99/)。 The genetic markers of the 7q1.1.23 region, which were found to have the strongest linkage obtained in Example 1 above, were examined for candidate genes adjacent to D7S2472 and D7S2415. As a result, the elastin gene was present (tit tp: //www.ncbi.nlm.nih.gov/genemap99/).
エラスチンは血管細胞外マトリックスの主要構成成分であるエラスチン線 維の中核をなす。 エラスチンは血管の弾性を担う分子であるとともに、 血管 の強化に関与していると考えられている（分子細胞生物学辞典 （東京化学同人 社発行， 1997年） 参照）。 脳動脈瘤の好発部位はウィルス輪などの脳血管 分岐部であり、 分岐部の脆弱性と脳動脈瘤発生との関連がこれまで指摘され ている（Carmichael, R. , J. Path. Bact. vol 62, 1-18 (1964) ；)。 連鎖解析 の結果、 及びエラスチンの機能的役割に関する公知の技術から、 エラスチン 遺伝子が脳動脈瘤の有力な疾患候補遺伝子であると考えられた。 実施例 3 エラスチン遺伝子の構造およびスクリーニング Elastin is at the core of the elastin fiber, 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 Cell Biology (Tokyo Kagaku Dojinsha, 1997)). The most common site of cerebral aneurysms 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);). As a result of linkage analysis, and from a known technique regarding the functional role of elastin, the elastin gene was considered to be a potential disease candidate gene for cerebral aneurysms. Example 3 Structure and Screening of Elastin Gene
エラスチン遺伝子は、 前記のように 7 Q 1 1. 23に存在し、 短腕テロメ
ァから' 8 6 . 5センチモルガン（cM)に位置する全長 5 5キロベースの 3 4個 のェキソンからなっている遺伝子である（Peoples, R.， et al. Am. J. Hum. Genet. 66 (1) 7-68 (2000))。 The elastin gene is located at 7Q11.23 as described above, A gene consisting of 34 exons with a total length of 55 kilobases located at 86.5 centiMorgans (cM) from the parent (Peoples, R., et al. Am. J. Hum. Genet. 66 (1) 7-68 (2000)).
このエラスチン遺伝子のすべてのェキソン、 プロモ一夕一領域、 そしてィ ントロンの一部について、 遺伝子変異のスクリーニングを直接シーケンス解 析にて行った（Rosenblmn, B. B. , et al . Nuc leic Ac ids Res. 25, 4500- 4504 (1997) ) For all exons, promo-overnight regions, and part of introns of this elastin gene, gene mutations were screened by direct sequence analysis (Rosenblmn, BB, et al. Nucleic Acids Res. 25 , 4500-4504 (1997))
その結果を表 2に示す。
The results are shown in Table 2.
一塩基 % (df = 1) ρ 位 置 s. ア ノ酸 アレル の頻度 Single base% (df = 1) ρ position s. Frequency of anoic acid allele
多型領域 変 異 患者 Polymorphic region mutation patient
ΡΜ 1 promoter (-1042) C→T 0.202 (77/382) 0.208 (69/332) 0.043 0.836ΡΜ 1 promoter (-1042) C → T 0.202 (77/382) 0.208 (69/332) 0.043 0.836
ΡΜ 2 promoter (-972) G→A 0.178 (68/382) 0.145 (48/332) 1.459 0.227ΡΜ 2 promoter (-972) G → A 0.178 (68/382) 0.145 (48/332) 1.459 0.227
ΡΜ 3 promoter (-38) C→T 0.021 (4/188) 0.040 (7/174) 1.102 0.294ΡΜ 3 promoter (-38) C → T 0.021 (4/188) 0.040 (7/174) 1.102 0.294
INT 4 intron 4 (+71) G→A 0.201 (76/378) 0.178 (60/338) 0.643 0.423INT 4 intron 4 (+71) G → A 0.201 (76/378) 0.178 (60/338) 0.643 0.423
EX 5 exon 5 (+61) C→T Ala-Val 0.021 (4/188) 0.029 (5/174) 0.207 0.649EX 5 exon 5 (+61) C → T Ala-Val 0.021 (4/188) 0.029 (5/174) 0.207 0.649
INT14 intron 14 (-28) G→A 0.016 (3/186) 0.035 (6/170) 1.324 0.250INT14 intron 14 (-28) G → A 0.016 (3/186) 0.035 (6/170) 1.324 0.250
EX 20 exon 20 (+114) G→A Gly-Ser 0.189 (71/376) 0.210 (71/338) 0.503 0.478EX 20 exon 20 (+114) G → A Gly-Ser 0.189 (71/376) 0.210 (71/338) 0.503 0.478
INT 20 intron 20 (+17) T→C 0.269 (101/376) 0.210 (71/338) 3.388 0.067INT 20 intron 20 (+17) T → C 0.269 (101/376) 0.210 (71/338) 3.388 0.067
INT 23 intron 23 (+24) T→C 0.294 (113/384) 0.308 (106/344) 0.166 0.684INT 23 intron 23 (+24) T → C 0.294 (113/384) 0.308 (106/344) 0.166 0.684
INT 27 intron 26 (-20) C→T 0.016 (3/192) 0.006 (1/174) 0.824 0.364INT 27 intron 26 (-20) C → T 0.016 (3/192) 0.006 (1/174) 0.824 0.364
INT 32 intron 32 (-34) C→T 0.052 (20/384) 0.056 (19/340) 0.051 0.821INT 32 intron 32 (-34) C → T 0.052 (20/384) 0.056 (19/340) 0.051 0.821
3UTR 1 3'UTR (+502) Aの挿入 0.102 (39/382) 0.144 (49/340) 2.968 0.0853UTR 1 3'UTR (+502) Insert A 0.102 (39/382) 0.144 (49/340) 2.968 0.085
3UTR 2 3'UTR (+659) G→C 0.050 (19/382) 0.060 (20/336) 0.153 0.6963UTR 2 3'UTR (+659) G → C 0.050 (19/382) 0.060 (20/336) 0.153 0.696
ΡΜ, promoter プロモーター ΡΜ, promoter
I NT, intron I NT, intron
Ε X, exon ：ェキソン
Ε X, exon: Exon
表 2から分かるように、 13ケ所で多型変異を検出した。 変異のうち、 ァ ミノ酸置換を起こす変異はェキソン 5 (61 番目 C→T:Ala→Val)とェキソン 2 0 (114番目 G— A:Gly→Ser)の 2ケ所のみであった。 その他の変異は、 イント ロン部位、 プロモータ一部位に存在していた。 遺伝子変異タイピングは、 一 塩基伸長反応を用いる SNaP s ho t法を AB I 377 DNAシーケンサ "(PE アプライドバイオシステムズ社製）を用い、 それぞれの変異でのアレル 頻度を検出した。 該方法は、 AB I 377 DNAアナライザーの使用マニュ アルに順じておこなった。 実施例 4 ケースコントロールスタディ As can be seen from Table 2, polymorphic mutations were detected at 13 sites. Among the mutations, only two mutations caused amino acid substitution: exon 5 (61st C → T: Ala → Val) and exon 20 (114th G—A: Gly → Ser). Other mutations were present at the intron site and at part of the promoter. Gene mutation typing was performed by using the SNaP short method using a single base extension reaction with ABI 377 DNA sequencer (manufactured by PE Applied Biosystems), and detecting the allele frequency at each mutation. Example 4 Case Control Study I 377 DNA Analyzer
エラスチン遺伝子多型と脳動脈瘤との関連性を調べるため、 実施例 3で得 られたすベての多型について、 ァレル頻度の比較をケ一スコントロールスタ ディでおこなった。 In order to examine the relationship between the elastin gene polymorphism and the cerebral aneurysm, allele polymorphisms obtained in Example 3 were compared for allele frequency using a case control study.
対象とした患者は、 アンギオグラフィー、 MR A (magnetic resonance angiography)又は 3 D C T A (3 dimentional computed t olographic angiography)のいずれかの検査により、 直径 4匪以上の脳動脈瘤を認めたも のであり、 同様の手法で脳動脈瘤が認められなかったものを非患者 (対照）と した。 患者 168名、 非患者 188名のそれぞれの多型について、 カイ検定 (X -テスト）を行い、 患者と対照とでアレル頻度の比較を行った。 The target patients had a cerebral aneurysm with a diameter of 4 or more by angiography, MRA (magnetic resonance angiography), or 3 DCTA (3 dimentional computed tolographic angiography). Non-patients (control) were those without cerebral aneurysm according to the method described above. Chi-tests (X-tests) were performed for each polymorphism in 168 patients and 188 non-patients, and allele frequencies were compared between patients and controls.
即ち、 ある遺伝子変異が病気の原因となっているならば、 患者と対照とで そのアレル頻度に差が予想される。 すべての多型でケ一スコントロールス夕 ディを行ったが、 特に頻度に差があるような多型はなかった。 そこで、 さら に二つの多型を組み合わせてハプロタイプ解析をおこない、 その頻度の比較 をおこなった。 尚、 ハプロタイプは A R L E Q U I Nプログラム (http://anthropologie.imige.ch/arlequin )により構築し、 可能な 2ケ所の 組み合わせ全ての解析をおこなった。 その結果を表 3に示す。
表 3 That is, if a gene mutation causes the disease, a difference in the allele frequency between the patient and the control is expected. Case control studies were performed for all polymorphisms, but none of the polymorphisms had a difference in frequency. Therefore, haplotype analysis was also performed by combining the two polymorphisms, and their frequencies were compared. The haplotype was constructed using the ARLEQUIN program (http://anthropologie.imige.ch/arlequin), and all possible combinations of two locations were analyzed. The results are shown in Table 3. Table 3
イントロン 20/イントロン 23 Intron 20 / Intron 23
, 患 ■ 患 非；τ、 患者 , Patient ■ Patient Non; τ, Patient
WW 0. 5577 0. 502 21 0 1 69 WV 0. 1737 0. 2867 65 96 VW 0. 1444 0. 1885 54 63 W 0. 1 242 0. 0229 47 8 WW 0.5557 0.502 21 0 1 69 WV 0.1737 0.2867 65 96 VW 0.1444 0.1885 54 63 W 0.1 242 0.0229 47 8
376 336 376 336
； C2 36. 62 C 2 36. 62
Ρ 5. 54 x 10-8
Ρ 5.54 x 10-8
その結果、 単独変異では有意な差はなかったが、 2ケ所を組み合わせるこ とにより有意な差を得ることが出来た。 最も強い有意差を得たのは、 イント ロン 2 0 ( + 1 7 ) とイントロン 2 3 ( + 2 4 ) のハプロタイプで、 値は P = 5 X 1 0— 8程度であった（表 3 )。 即ち、 イントロン 2 0とイントロン 2 3 多型は、 単独では疾患に関与していないが、 両者が組み合わさることにより、 疾患に関与していることが判明した。 As a result, there was no significant difference in the single mutation, but a significant difference could be obtained by combining the two sites. Strongest give a significant difference, in haplotypes intron 2 0 (+ 1 7) and intron 2 3 (+ 2 4), the value was about P = 5 X 1 0- 8 (Table 3) . That is, it was found that intron 20 and intron 23 polymorphism alone were not involved in the disease, but that both were involved in the disease when combined.
従って、 これらイントロン 2 0とイントロン 2 3多型を有するものが、 脳 動脈瘤の発症リスクを保有するヒトまたは患者としての確率が高いことにな る。 即ち、 エラスチン遺伝子のイントロン 2 0の 1 7番目の野生型 Tであり、 イントロン 2 3の 2 4番目の Tが Cに変異したハプロタイプを有するヒトが 脳動脈瘤の発症リスクが高いことがわかる。 実施例 5 脳動脈瘤の発症リスクの検出法 Therefore, those having these intron 20 and intron 23 polymorphisms have a high probability of being a human or patient having a risk of developing a cerebral aneurysm. In other words, it is understood that a human having a haplotype in which the 17th intron 20 of elastin gene is intron 20 and the 24th T of intron 23 is mutated to C has a high risk of developing a cerebral aneurysm. Example 5 Method for detecting risk of developing cerebral aneurysm
1 . エラスチン遺伝子のイントロン 2 0とイントロン 2 3ハプロタイプの 患者において有意に頻度が高かったのは、 WV (イントロン 2 0は野生型 (W)で、 イントロン 2 3は変異型（V) ) であった （これを WVハプロタイプ とする。 ） 。 1. Significantly higher frequency in patients with the intron 20 and intron 23 haplotypes of the elastin gene was WV (intron 20 was wild-type (W) and intron 23 was mutant (V)). (This is called the WV haplotype.)
WVハプロタイプに注目すると、 相対危険率（odds rat io)は、 1 . 9 1、 即ち、 このハプロタイプを有しているヒトは、 このハプロタイプを有してい ないヒトより 1 . 9倍も脳動脈瘤に罹患しやすいということである。 Focusing on the WV haplotype, the relative risk (odds rat io) is 1.91, ie, humans with this haplotype are 1.9 times more cerebral aneurysms than those without this haplotype Susceptible to the disease.
また WVタイプのハプロタイプをホモ接合性で有しているヒトは、 脳動脈 瘤で有意に多く （10. 7% vs. 2. 7¾, P=0. 002) 、 相対危険率は 4 . 3 9であつ た。 すなわち WVタイプをホモ接合性で有する個人は脳動脈瘤の発症リスク が非常に高いことが示唆される。 これは一般に以下のように計算される。 In addition, those who have homozygous WV-type haplotypes are significantly more likely to have cerebral aneurysms (10.7% vs. 2.7¾, P = 0.002), and have a relative risk of 4.3 9 It was. This suggests that individuals who are homozygous for the WV type have a very high risk of developing a cerebral aneurysm. This is generally calculated as follows:
患者 対照 Patient control
変異アレル （WV) a c Mutant allele (WV) a c
野生型アレル （その他） b d 相対危険率 = a X d / b X c
このような結果によっても、 ヒトのエラスチン遺伝子のタイピングが脳動 脈瘤発症リスクの存在の診断的な意義を有するものと考えられた。 Wild-type allele (other) bd Relative risk = a X d / b X c These results suggest that human elastin gene typing has diagnostic significance for the risk of developing cerebral aneurysms.
また、 この遺伝型は脳動脈瘤破裂（クモ膜下出血)患者で増えるということ はなかったので、 脳動脈瘤破裂ではなく脳動脈瘤発生そのものに関与するも のと予想された。 In addition, since this genotype did not increase in patients with cerebral aneurysm rupture (subarachnoid hemorrhage), it was expected that this genotype would be involved in cerebral aneurysm development itself rather than cerebral aneurysm rupture.
従って、 脳動脈瘤の発症リスクの存在を検出するためには、 イントロン 2 0 (17番目の T→C)とイントロン 23 (24番目の T→C)の 2ケ所それぞ れを測定対象として、 遺伝子タイピングする必要がある。 この 2ケ所からハ プロタイプを構築し、 イントロン 20の 17番目の DN Aが野生型 Tでイン トロン 23の 24番目 Tが Cであるハプロタイプを検出する必要がある。 Therefore, in order to detect the risk of developing a cerebral aneurysm, two locations, intron 20 (17th T → C) and intron 23 (24th T → C), were measured. 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.
該方法としては、 1塩基伸長反応をおこなうスナップショット（SNa P s h o t法）が好ましく例示される。 該方法は、 AB I PR I SM SN a P s h o t d dNTP P r ime r Ex t en t i on K i t (ABI ノ ィォシステムズ社製）にて簡便に測定できる。 反応後生成した蛍光フラグメン トは、 同じく AB I a PR I SM 310ノ 377Z3100/3700お よび Ge n e S c a nソフトウェアで解析することができる。 As the method, a snapshot (SNaP shot method) in which a single base extension reaction is performed is preferably exemplified. This method can be simply measured by using ABPIPRISMSNaPshotddNTPPrimerExtensionKit (manufactured by ABI Nano Systems). Fluorescence fragments generated after the reaction can be analyzed with ABI PRIMSM310NO377Z3100 / 3700 and GeneScan software as well.
即ち、 対象者から得られた検体からゲノム DNAを抽出した後、 ヒト 'ェ ラスチン遺伝子のイントロン 20およびイントロン 23の部分の DN A断片 をそれぞれ含むように作成したプライマー対によって、 数十から数百塩基の DNA断片長を P CR増幅する。 この P CR産物をテンプレートにして、 目 的の変異 DN Aの直前に対応するプライマーをァニーリングさせ、 ポリメラ —ゼで 1塩基のみ伸長させる。 例えばイントロン 20の場合、 アンチセンス 鎖にハイプリダイズさせる塲合は、 イントロン 20部位の D N A配列番号 1 6番目からセンス鎖の 5' 側に任意の長さ， 例えば約 16— 24塩基長の野 生型配列からなるプライマーを作成する。 センス鎖にハイブリダィズさせる 場合にはィントロン 20部位の D N A配列番号 18番目からァンチセンス鎖 の 5 ' 側に任意の長さ， 例えば約 16— 24塩基長の野生型配列からなるプ ライマー、 例えばイントロン 20の DNA配列 （配列番号 1) 中番号 1 8〜 37の配列に相補的な 20塩基長の核酸配列からなるプライマー （配列番号
3) を合成する。 そして 6—F AMと HEXなどの ATGCのそれぞれ異な つた色の蛍光でラベルされた d d NT Pを混ぜておく。 d dNTPは 1塩基 の伸長で伸長がストップする塩基であるから、 この反応の結果により 1塩基 のみ伸長したプライマーは、 伸長した端の塩基の種類に依存して色が着く。 即ち、 イントロン 20部位の DNA配列の 17番目が野生型か変異型かを判 別することが出来る。 同様にイントロン 23部位の 24番目の DN Aが野生 型か変異型かを判別することが出来る。 例えば、 イントロン 23の DNA配 列 （配列番号 2) 中番号 25〜44の配列に相補的な 20塩基長の核酸配列 からなるプライマ一 （配列番号 4) を合成して上記と同様に判別する。 That is, after extracting genomic DNA from a sample obtained from a subject, several tens to several hundreds of primer pairs were prepared to contain the DNA fragments of the intron 20 and intron 23 portions of the human elastin gene, respectively. PCR amplifies the base DNA fragment length. Using this PCR product as a template, anneal the corresponding primer immediately before the target mutant DNA, and extend only one base with polymerase. For example, in the case of intron 20, the nucleotide to be hybridized to the antisense strand is a wild-type of any length, for example, about 16 to 24 nucleotides in length from the DNA sequence No. 16 at the intron 20 site to the 5 'side of the sense strand. Create a primer consisting of the type sequence. When hybridizing to the sense strand, a primer consisting of a wild-type sequence of an arbitrary length, for example, about 16 to 24 bases in length from the DNA sequence number 18 at the intron 20 to the 5 'side of the antisense strand, such as intron 20, is used. DNA sequence (SEQ ID NO: 1) Primer consisting of a nucleic acid sequence of 20 bases complementary to the sequence of Nos. 18 to 37 (SEQ ID NO: 1) 3) is synthesized. Then, mix 6-FAM and ddNTP labeled with different colors of fluorescence of ATGC such as HEX. Since d dNTP is a base that stops elongating by one base extension, the primer that has extended only one base will have a color depending on the type of base at the extended end as a result of this reaction. That is, it can be determined whether the 17th DNA sequence of the 20 introns is a wild type or a mutant type. Similarly, it is possible to determine whether the 24th DNA of intron 23 is wild type or mutant type. For example, a primer (SEQ ID NO: 4) consisting of a 20-base long nucleic acid sequence complementary to the sequence of ID Nos. 25 to 44 in the DNA sequence of Intron 23 (SEQ ID NO: 2) is synthesized and discriminated in the same manner as described above.
この d dNTPによって蛍光ラベルされたプライマーを電気泳動で分離し、 蛍光シグナルを解析検出する。 尚、 蛍光シグナルを電気泳動で検出する方法 に代えて、 1塩基伸長の伴った分子量の差をマススぺクトルで検出する方法 も適用することができる。 The primers fluorescently labeled with this ddNTP are 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 with a mass spectrum can also be applied.
このように変異によって取り込まれる塩基が異なり、 異なる蛍光シグナル として認識されることから、 遺伝子タイピングが可能となる。 かくして、 1 96サンプル（5 ngDN Aを P CR増幅）を 1枚のゲル（泳動 2時間）で解析で さる。 In this way, the base incorporated by the mutation is different and is recognized as a different fluorescent signal, so that genotyping is possible. Thus, 196 samples (PCR amplification of 5 ng DNA) are analyzed on a single gel (2 hours running).
3についても同様に検出できる。 実施例 5— 2 脳動脈瘤の発症リスクの検出法 3 can be similarly detected. Example 5-2 Method for Detecting Risk of Onset of Cerebral Aneurysm
ヒロシーケンス (Pyrosequencing： Ronaghi, M. , et al. , Science, 281, 363- 365 (1998))法により行った。 即ち、 血液サンプルなどから、 常法により ゲノムを単離した後、 ピオチン標識したプライマーにより変異を含むターゲ ットの数十から数百塩基を P CR増幅して行った。 マグネットビーズを用い、 一本鎖 D N Aを精製し、 変異の数塩基上流からシーケンスするように設定さ れたプライマーをァニーリングさせた。 次にソフトウェアに入力された変異 付近のシーケンスに従って、 装置に 1種類ずつ dNTPを添加した。 DNA ポリメラーゼが塩基伸長すると PP i (ピロリン酸） が生成した。 ピロリン 酸（PP i)をスルフリラーゼ（Sulfurylase)により ATPに返還させ、 それを
ルシフェラ一ゼの基質とし、 発光検出器や C C Dカメラで化学発光を検出し た。 添加した dNTP、 即ち、 塩基配列に従い、 発光のピークが得られるの で、 遺伝子タイピングが可能となる。 この手法により 96サンプルを 15分 ほどでタイピングできた。 This was performed by the method of Hirosequencing (Pyrosequencing: Ronaghi, M., et al., Science, 281, 363-365 (1998)). That is, after a genome was isolated from a blood sample or the like by a conventional method, PCR was performed to amplify tens to hundreds of bases of a target containing a mutation using a biotin-labeled primer. Single-stranded DNA was purified using magnet beads, and primers designed to sequence from several bases upstream of the mutation were annealed. Next, dNTPs were added to the device one by one according to the sequence near the mutation input to the software. When the DNA polymerase base-extended, PP i (pyrophosphate) was generated. Pyrophosphate (PP i) is returned to ATP by sulfurylase, Chemiluminescence was detected using a luminescence detector or CCD camera as a substrate for luciferase. Since a luminescence peak is obtained according to the added dNTP, that is, the nucleotide sequence, genotyping becomes possible. With this method, 96 samples could be typed in about 15 minutes.
上記方法は、 DNAポリメラーゼ、 ATP—スルフリラ一ゼ、 ルシフェラ —ゼ、 ァピラーゼ（apyrase)の 4種の酵素混合液、 ルシフェリン、 APS (ァ デノシン 5' 硫酸リン酸） からなる基質液、 d AT P (デォキシアデノシン α ーチォ · 3リン酸）、 dCTP、 dGTP、 d T T Pからなる d NT Pを構成 要素とする市販の S NPR e ag en t K i t s (Pyrosequencing AB社製） を用い、 該方法を用いる自動 DNA配列分析のための P SQ 96システム (Pyrosequencing AB 社製）およびその使用のための S NPソフトウェア (Pyrosequencing AB社製）を用いて、 簡便に測定することができる。 The above method is a substrate solution consisting of DNA enzyme, ATP-sulfurylase, luciferase, four kinds of enzyme mixture of apyrase, luciferin, APS (adenosine 5 'sulfate phosphate), d ATP ( Using commercially available SNPR eag ent K its (manufactured by Pyrosequencing AB) having dNTP consisting of doxyadenosine α-thio-3-phosphate), dCTP, dGTP and dTTP as components, The measurement can be carried out simply using a P SQ 96 system (manufactured by Pyrosequencing AB) for automatic DNA sequence analysis and SNP software (manufactured by Pyrosequencing AB) for use.
このように、 本発明によって見出されたヒト ·エラスチン遺伝子のイント ロン 20とイントロン 23の WV八プロタイプを有するものが、 脳動脈瘤の 発症リスクが高いという事実から、 該方法を用いて、 簡便に、 ヒトの脳動脈 瘤発症リスクの存在を検出することが可能となる。 Thus, the fact that the human elastin gene intron 20 and intron 23 WV eight prototypes found by the present invention has a high risk of developing a cerebral aneurysm, It is possible to easily detect the presence of human cerebral aneurysm development risk.
また、 本発明の脳動脈瘤発症リスク検出法の利用によって、 脳動脈瘤の遺 伝的なリスクを有するヒトを同定し、 出血する前に診断する、 二次予防の体 制確立が可能となる。 また、 本発明の利用によって、 脳ドックや手術の対象 者を適切に選定することも可能となる。 In addition, by using the method for detecting a risk of developing a cerebral aneurysm according to the present invention, it is possible to establish a secondary prevention system in which a person having a genetic risk of a cerebral aneurysm is identified and diagnosed before bleeding. . Further, by using the present invention, it is also possible to appropriately select a target for a brain dock or an operation.
さらに本発明を利用して原因遺伝子がどのように脳動脈流疾患の発症へ関 与しているかを解析することにより、 脳動脈瘤成因解明が可能となり、 病態 生理に基づいた新たな脳動脈瘤（クモ膜下出血）に対する薬剤の開発なども可 能となる。 産業上の利用の可能性 Furthermore, by analyzing how the causative gene is involved in the development of cerebral artery flow disease using the present invention, it becomes possible to elucidate the causes of cerebral aneurysms, and to develop new cerebral aneurysms based on pathophysiology. (Subarachnoid hemorrhage) can also be developed. Industrial applicability
本発明によれば、 ヒ卜脳動脈瘤の発症のリスクに関連する遺伝子ハプロタ イブが提供される。 According to the present invention, there is provided a gene haplotype associated with a risk of developing a human cerebral aneurysm.
本発明の遺伝子八プロタイプは、 ヒト脳動脈瘤の発症においてその連鎖が
強く、 これら細胞及びその細胞周辺組織における脳動脈血管、 特にクモ膜下 出血の発症を促す因子と考えられる'。 本発明におけるヒト ·エラスチン遺伝 子のイントロン 2 0における特定部位の遺伝子多型およびイントロン 2 3·に おける特定部位の遺伝子多型の解析は、 遺伝子の機能と脳動脈瘤発症との係 わりに関する研究に利用でき、 特にヒトまたは脳動脈瘤（クモ膜下出血)患者 への遺伝子診断、 二次予防、 脳動脈瘤（クモ膜下出血）の再発予測、 手術の適 応及び該変異遺伝子に対する野生型 D N A鎖による医薬用途への研究に応用 することが可能である。 The gene eight prototype of the present invention has a linkage in human cerebral aneurysm development. It is strongly considered to be a factor that promotes the development of cerebral artery blood vessels in these cells and their surrounding tissues, especially subarachnoid hemorrhage. ' In the present invention, the analysis of the polymorphism at a specific site in intron 20 of the human elastin gene and the polymorphism at a specific site in intron 23 · is a study on the relationship between gene function and onset of cerebral aneurysm. In particular, it can be used for human or cerebral aneurysm (subarachnoid haemorrhage) genetic diagnosis, secondary prevention, recurrence prediction of cerebral aneurysm (subarachnoid haemorrhage), adaptation for surgery, and wild-type for the mutant gene It can be applied to research for medical use by DNA chain.
更に、 本発明によれば、 ヒト，エラスチン遺伝子のイントロン 2 3部位の 野生型オリゴヌクレオチドを含有する、 遺伝子治療に有用な遺伝子導入用べ クタ一、 該ヒトエラスチン遺伝子のイントロン 2 3部位の野生型オリゴヌク レオチドを導入した細胞、 該ベクター又は細胞を有効成分とする遺伝子治療 剤、 及びその利用による遺伝子治療法なども提供される。 Further, according to the present invention, there is provided a gene transfer vector useful for gene therapy, comprising a wild-type oligonucleotide at the intron 23 site of the human elastin gene, a wild-type at the intron 23 site of the human elastin gene. Also provided are a cell into which an oligonucleotide has been introduced, a gene therapy agent comprising the vector or the cell as an active ingredient, and a gene therapy method utilizing the same.
また、 本発明によれば、 ヒト ·エラスチン遺伝子のイントロン 2 0の特定 部位の遺伝子多型およびイントロン 2 3の特定部位の遺伝子多型の八プロ夕 イブを原因とする脳動脈瘤（クモ膜下出血）発症に関し、 そのリスクを低下さ せるような候補化合物のスクリーニング方法、 及びスクリーニング用キット を提供することもできる。
In addition, according to the present invention, a cerebral aneurysm (subarachnoid) caused by a polymorphism at a specific site of intron 20 and a polymorphism at a specific site of intron 23 of the human elastin gene is caused by octaprobe. It is also possible to provide a screening method of a candidate compound that reduces the risk of the occurrence of (bleeding), and a screening kit.
Claims
1 . ヒト ·エラスチン遺伝子のイントロンの部分の遺伝子多型を同定する ことにより、 脳動脈瘤の発症のリスクの存在を判定する方法。 1. A method for determining the risk of developing a cerebral aneurysm by identifying genetic polymorphisms in the intron portion of the human elastin gene.
2 . ヒト 'エラスチン遺伝子における、 2 0番目のイントロン部位と 2 3 番目のイントロン部位の遺伝子多型を同定することにより、 脳動脈瘤の発症 のリスクの存在を判定する請求の範囲 1に記載の方法。 2. The method according to claim 1, wherein the presence of a risk of cerebral aneurysm is determined by identifying a genetic polymorphism at the 20th intron site and a 23rd intron site in the human 'elastin gene. Method.
3 . ヒト ·エラスチン遺伝子の 2 0番目のイントロン部位 D N A配列の 1 7番目の D N Aが野生型 （T) であって、 且つ 2 3番目のイントロン部位 D N A配列の 2 4番目の D N Aが野生型 （T) から （C) に変異した変異型で ある遺伝子多型を同定することにより、 脳動脈瘤の発症のリスクの存在を判 定する請求の範囲 1又は 2のいずれかに記載の方法。 3. The 17th DNA of the 20th intron DNA sequence of the human elastin gene is the wild type (T), and the 24th DNA of the 23rd intron DNA sequence is the wild type (T). 3. The method according to claim 1, wherein the presence of a risk of cerebral aneurysm development is determined by identifying a gene polymorphism that is a variant mutated from T) to (C).
4 . 遺伝子多型を、 ヌクレオチド直接配列決定法、 対立遺伝子特異的オリ ゴヌクレオチド（AS0)—ドットプロット分析、 一塩基プライマー伸長法、 P C R—単鎖高次構造多型（SSCP)分析、 P C R—制限酵素断片長多型 (RFLP)分析、 インベーダー法および定量的リアルタイム P C R検出法からなる群から選ば れる少なくとも一つの方法を用いて同定する請求の範囲 1乃至 3のいずれか に記載の方法。 4. Genetic polymorphisms can be analyzed by direct nucleotide sequencing, allele-specific oligonucleotide (AS0)-dot plot analysis, single-base primer extension, PCR-single-stranded conformation polymorphism (SSCP) analysis, PCR- 4. The method according to any one of claims 1 to 3, wherein the identification is performed using at least one method selected from the group consisting of restriction enzyme fragment length polymorphism (RFLP) analysis, invader method, and quantitative real-time PCR detection method.
5 . 一塩基プライマ一伸長法を用いて同定する請求の範囲 4に記載の方法。 5. The method according to claim 4, wherein the method is identified using a single nucleotide primer-extension method.
6 . 以下の （a ) 及び （b ) に記載のオリゴヌクレオチドの組み合わせか らなるプライマー又はプローブ。 6. A primer or probe comprising a combination of the oligonucleotides described in (a) and (b) below.
( a ) ヒト ·エラスチン遺伝子にハイブリダィズすることができる遺伝子多 型検出用プライマー又はプローブとしてのオリゴヌクレオチドであって、 該 オリゴヌクレオチドが、 ヒト ·エラスチン遺伝子の 2 0番目のイントロン部
位 DNA配列の 17番目の DN Aの遺伝子多型部位を含む配列を有するか、 又は、 該オリゴヌクレオチドの 3 ' 末端が該遺伝子多型部位よりも 1塩基か ら数塩基上流に位置するものであるオリゴヌクレオチド、 (a) an oligonucleotide as a primer or probe for detecting a gene polymorphism capable of hybridizing to the human elastin gene, wherein the oligonucleotide is an intron at position 20 of the human elastin gene The DNA sequence has a sequence containing the 17th DNA polymorphic site of the DNA sequence, or the 3 'end of the oligonucleotide is located one to several bases upstream from the polymorphic site. An oligonucleotide,
(b) ヒト ·エラスチン遺伝子にハイブリダィズすることができる遺伝子多 型検出用プライマー又はプローブとしてのオリゴヌクレオチドであって、 該 オリゴヌクレオチドが、 ヒト ·エラスチン遺伝子の 23番目のイントロン部 位 D N A配列の 24番目の D N Aの遺伝子多型部位を含む配列を有するか、 又は、 該オリゴヌクレオチドの 3' 末端が該遺伝子多型部位よりも 1塩基か ら数塩基上流に位置するものであるオリゴヌクレオチド。 (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 24th intron DNA sequence of the 23rd intron of the human elastin gene. Or an oligonucleotide having a sequence containing the gene polymorphic site of the above DNA, or having the 3 ′ end of the oligonucleotide located one to several bases upstream from the gene polymorphic site.
7. ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DNA配列の 1 7番目の DNAの遺伝子多型検出用プライマー、 及び、 ヒト ·エラスチン遺 伝子の 23番目のイントロン部位 DN A配列の 24番目の DN Aの遺伝子多 型検出用プライマーを含む脳動脈瘤の発症のリスク検出用診断キット。 7. Intron at position 20 in the human elastin gene Primer for detecting a polymorphism in the DNA at position 17 in the DNA sequence, and intron 23 in the human elastin gene DN24 in the DNA sequence A diagnostic kit for detecting the risk of developing cerebral aneurysms, comprising a primer for detecting the gene polymorphism A.
8. ヒト ·エラスチン遺伝子の 20番目のイントロン部位 DNA配列の 1 7番目の DNAが野生型 （T) であって、 且つ 23番目のイントロン部位 D NA配列の 24番目の DNAが野生型 （T) から （C) に変異した変異型で ある、 脳動脈瘤の発症のリスク検出のための DN A配列からなる八プロタイ プ及びその相補鎖。
8. The 17th DNA in the 20th intron DNA sequence of human elastin gene is wild type (T) and the 24th DNA in the 23rd intron DNA sequence is wild type (T) Eight protypes comprising a DNA sequence for detecting the risk of developing a cerebral aneurysm and their complementary strands, which are mutated from (C) to (C).
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| JP2001042907A JP2002238577A (en) | 2001-02-20 | 2001-02-20 | Cerebral aneurysm-sensitive gene |
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| RU2627643C2 (en) * | 2015-08-18 | 2017-08-09 | Государственное бюджетное учреждение "Академия наук Республики Саха(Якутия)" (ГБУ АН РС(Я) | Method for prediction of risk of subarachnoidal bleeding due to brain vessels aneurism rupture in persons of asian race |
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| JP5128796B2 (en) * | 2006-01-20 | 2013-01-23 | Ｇ＆Ｇサイエンス株式会社 | Genetic risk detection method for cerebrovascular disorders |
| JP5695383B2 (en) | 2009-10-05 | 2015-04-01 | 花王株式会社 | Hair shape susceptibility gene |
| JP5695384B2 (en) | 2009-10-05 | 2015-04-01 | 花王株式会社 | Hair shape susceptibility gene |
| JP5695385B2 (en) | 2009-10-05 | 2015-04-01 | 花王株式会社 | Hair shape susceptibility gene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2627643C2 (en) * | 2015-08-18 | 2017-08-09 | Государственное бюджетное учреждение "Академия наук Республики Саха(Якутия)" (ГБУ АН РС(Я) | Method for prediction of risk of subarachnoidal bleeding due to brain vessels aneurism rupture in persons of asian race |
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