WO2010073544A1 - Méthode de détermination du risque de maladies coronariennes - Google Patents

Méthode de détermination du risque de maladies coronariennes Download PDF

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WO2010073544A1
WO2010073544A1 PCT/JP2009/006918 JP2009006918W WO2010073544A1 WO 2010073544 A1 WO2010073544 A1 WO 2010073544A1 JP 2009006918 W JP2009006918 W JP 2009006918W WO 2010073544 A1 WO2010073544 A1 WO 2010073544A1
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mkl1
subject
coronary artery
artery disease
detecting
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Japanese (ja)
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木村彰方
日野原邦彦
中島敏晶
和泉徹
猪子英俊
山田芳司
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国立大学法人 東京医科歯科大学
学校法人北里研究所
学校法人東海大学
国立大学法人三重大学
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Priority to JP2010543810A priority Critical patent/JPWO2010073544A1/ja
Publication of WO2010073544A1 publication Critical patent/WO2010073544A1/fr

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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention relates to a method for examining a subject's risk for coronary artery disease.
  • Coronary artery disease is one of the important health problems in developed countries due to its increased morbidity and high mortality. Coronary artery disease is mainly caused by atheroma of coronary artery inner wall containing lipids mainly composed of cholesterol ester, mucopolysaccharide, fibrous tissue and the like.
  • the lesion includes smooth muscle cells and macrophages that are foamed with a large amount of fat droplets as cell components.
  • As the atheroma rises in the coronary arteries surrounding the heart the lumen is narrowed and blood flow is partially blocked. As a result, there is a shortage of blood supplied to the myocardium, causing myocardial ischemia, and the heart cannot normally contract and pump blood. Myocardial necrosis can also occur when the atheroma completely blocks the artery.
  • Reversible ischemia in the coronary arteries is angina and irreversible ischemia is myocardial infarction, causing sudden chest pain.
  • Non-Patent Document 1 Although environmental factors such as smoking, hypertension, hypercholesterolemia, and diabetes are deeply involved in the onset of coronary artery disease (see, for example, Non-Patent Document 1), genetic factors also play an important role. There is also evidence to show that this is present (for example, see Non-Patent Document 2). Accordingly, it is generally recognized that both environmental and genetic factors are associated with the pathogenesis of coronary artery disease.
  • an object of the present invention is to provide a method for examining coronary artery disease using an SNP or the like that has been sufficiently verified to be associated with a disease, a reagent used in the method, and the like.
  • cytosine (C) at position ⁇ 184 in the promoter region of megakaryoblastic leukemia factor-1 (hereinafter referred to as “MKL1”) gene is thymine.
  • the SNP replaced with (T) is highly related to the prevalence of coronary artery disease; if the SNP is present, the promoter activity of the MKL1 gene is enhanced; if the SNP is present, the SNP is against the promoter of the MKL1 gene Reduced affinity of the nuclear factor, which appears to be a suppressor, to the promoter; increased expression of MKL1 protein in foamed macrophages in atherosclerotic lesions; based on these facts, the MKL1 gene was strongly suggested to be one of the pathogenic genes of coronary artery disease, and the present invention was completed.
  • a method for examining the risk of coronary artery disease in a subject comprising a step of detecting a mutation that increases MKL1 gene expression or an increase in MKL1 gene expression in a sample collected from the subject; [2]
  • the step of detecting a mutation that enhances the expression of the MKL1 gene includes a step of detecting a polymorphism at a site corresponding to position 817 of the base sequence described in SEQ ID NO: 1 in the MKL1 gene.
  • a test agent for coronary artery disease risk comprising at least one of the following (1) to (3): (1) A nucleic acid that hybridizes to a nucleic acid consisting of a region of at least 10 bases including position 817 in the base sequence shown in SEQ ID NO: 1 or a nucleic acid consisting of a region complementary thereto (2) described in
  • a method for diagnosing coronary artery disease risk in a subject comprising a step of detecting a mutation that increases MKL1 gene expression or an increase in MKL1 gene expression in a sample collected from the subject;
  • the step of detecting a mutation that enhances MKL1 gene expression in a sample collected from the subject includes a step of preparing a nucleic acid sample from a sample collected from the subject, and a sequence in the MKL1 gene in the nucleic acid sample.
  • Detecting the polymorphism of the site corresponding to position 817 of the base sequence described in No. 1 the method according to [7] above; [9] The method according to [8] above, wherein when the polymorphism in which the base corresponding to the position 817 is thymine (T) is detected, the subject is diagnosed with a higher risk of coronary artery disease than usual. ; [10] The step of detecting the increase in MKL1 gene expression in the sample collected from the subject includes the step of collecting a sample containing activated macrophages from the subject, and the expression of the MKL1 gene in the activated macrophages.
  • a method of detecting the enhancement of [11] The method according to [10] above, wherein when the expression of the MKL1 gene is enhanced, the subject is diagnosed with a coronary artery disease risk higher than usual; [12] A method for screening a prophylactic or therapeutic agent for coronary artery disease, comprising a step of contacting a cell capable of expressing the MKL1 gene with the prophylactic or therapeutic drug candidate, and the expression of the MKL1 gene or the function of the MKL1 protein Selecting a candidate that inhibits or suppresses; [13] DNA encoding the MKL1 protein consisting of a base sequence in which the base corresponding to position 817 in the base sequence described in SEQ ID NO: 1 is T; [14] A recombinant vector comprising the DNA of [13] above; [15] A transformant containing the recombinant vector according to [14] above.
  • a subject is diagnosed with coronary artery disease in a subject by detecting the presence or absence of a mutation that increases MKL1 gene expression or the presence or absence of an increase in MKL1 gene expression. Presence / absence and risk of coronary artery disease can be determined.
  • the SNP at the position corresponding to position 817 of the base sequence described in SEQ ID NO: 1 in the MKL1 gene has been confirmed by a replication study and is sufficiently related to the disease. It has been verified.
  • the coronary artery disease risk of the subject can be easily determined.
  • the onset of coronary artery disease depends on environmental factors as well as genetic factors. Therefore, when it is determined by the method according to the present invention that the risk of coronary artery disease due to genetic factors is high, the onset can be suppressed by suppressing environmental factors.
  • a preventive or therapeutic agent for coronary artery disease can be identified.
  • administering the prophylactic or therapeutic agent it is possible to reduce the expression level of the MKL1 gene, which is considered to be one of the causes of coronary artery disease, or to suppress the function of the MKL1 protein.
  • FIG. 1 shows an outline of the procedure for genome-wide screening.
  • FIG. 2 shows the results of genome-wide screening.
  • FIG. 3 shows the position of the MS marker CAD1-MS on chromosome 22q13.
  • FIG. 4 shows the results of expression analysis. 4A and 4B show the results of a reporter assay using K562 cells and HeLa cells, respectively. In any cell, it was confirmed that the luciferase activity driven by the sensitive allele T was higher than the luciferase activity driven by the allele C.
  • FIG. 4C shows the result of quantitative real-time PCR comparing the expression level of the MKL1 gene in a B cell line having a T allele homozygote and a B cell line having a C allele homozygote.
  • FIG. 5 shows the results of the gel shift assay.
  • FIG. 5A shows that the DNA-protein complex exhibits a higher affinity for -184C than for -184T.
  • FIG. 5B shows that the efficiency of competition with unlabeled DNA is higher when the -184C probe is included than when the -184T probe is included.
  • FIG. 6 shows the results of immunohistochemical staining.
  • FIG. 6A shows that MKL1 protein is expressed in the core of atherosclerotic plaque.
  • FIG. 6B shows the results of staining adjacent regions with anti-CD68 antibody. It was confirmed that the MKL1-positive cells were foamed macrophages.
  • FIG. 6C shows the result of staining with anti-human smooth muscle ⁇ -actin monoclonal antibody. It was confirmed that the MKL1 protein was also expressed in smooth muscle ⁇ -actin positive cells in the neointimal.
  • the method for examining coronary artery disease risk includes a step of detecting a mutation that increases MKL1 gene expression or an increase in MKL1 gene expression in a sample collected from a subject.
  • the coronary artery disease risk test method refers to a method for determining the high possibility of having coronary artery disease for a subject.
  • the inspection method according to the present invention can be implemented by, for example, an inspection company.
  • the first aspect of the method for examining coronary artery disease risk includes a step of detecting a mutation that enhances MKL1 gene expression in a sample collected from a subject.
  • the mutation that enhances the expression of the MKL1 gene may be any mutation, and includes mutations at the DNA level, transcription level, and translation level.
  • a site corresponding to position 817 of the base sequence described in SEQ ID NO: 1 It is preferable to detect a polymorphism at position M-184.
  • this method will be specifically described.
  • the test method according to the present invention not only examines and diagnoses the risk that a subject who does not currently suffer from coronary artery disease will suffer from the future, but also examines whether or not he / she currently suffers from coronary artery disease. It can also be used for diagnosis.
  • the “sample collected from the subject” may be any sample as long as the polymorphism of the MKL1 gene can be detected.
  • DNA may be isolated from these samples to detect the polymorphism, or RNA can be isolated and detected. It can also be detected using cDNA prepared from RNA.
  • the method for isolating DNA or RNA from a sample is not particularly limited, and chromosomal DNA or RNA can be extracted and isolated by methods known to those skilled in the art.
  • the method for detecting the polymorphism is not particularly limited, and various methods known to those skilled in the art can be employed. For example, the following methods can be used.
  • PCR-RFLP Restriction Fragment Length Polymorphism
  • a gene having a specific polymorphism is a method for detecting a polymorphism by utilizing the fact that the lengths of fragments produced by cleavage with a restriction enzyme are different.
  • a region containing the polymorphism to be detected is amplified by the PCR method.
  • a restriction enzyme that recognizes the polymorphic site is allowed to act on the amplified product, and the fragments are separated and identified by electrophoresis or the like. From the length of the generated fragment, it is possible to confirm the presence or absence of restriction enzyme digestion and thus the presence or absence of polymorphism.
  • cDNA can be obtained by reverse transcriptase and used instead of chromosomal DNA.
  • Allele-specific PCR When PCR is performed using a primer that hybridizes to the region containing the polymorphism (allele-specific oligonuceoide: ASO) and there is a polymorphism, mismatch occurs between the primer and the template DNA, so the annealing temperature is high.
  • ASO allele-specific oligonuceoide
  • the presence or absence of polymorphism in the sample DNA can be known by separating and identifying the amplification product by electrophoresis or the like and confirming the presence or absence of amplification.
  • chromosomal DNA obtained from a subject is amplified by PCR using a primer labeled with 32 P or biotin.
  • the obtained labeled DNA fragment is denatured by heating to give a single-stranded DNA, which is separated by polyacrylamide gel electrophoresis, and a change in the position of the band is detected by autoradiography.
  • DGGE Denaturing gradient gel electrophoresis
  • the gel is dissociated into a single strand at a lower concentration of the denaturing agent, so that the migration speed becomes slower in the gel region where the denaturing agent concentration is low. Therefore, by comparing with the band of the normal DNA sample, it is possible to detect the presence or absence of mismatch, and as a result, the presence or absence of the polymorphism can be detected.
  • concentration gradient of the denaturant may be applied vertically (vertical gradient method) or in parallel (parallel gradient method).
  • TGGE temperature gradient gel electrophoresis
  • (v) Method using microarray This is a method for detecting the presence of polymorphism by detecting specific hybridization between probe DNA immobilized on a microarray and a DNA or RNA sample.
  • a method for detecting the presence or absence of hybridization hybridization is performed by matching the 3 'end of the probe DNA with the expected polymorphic site, and labeled dideoxynucleotide and DNA polymerase are added to detect the presence or absence of an extension reaction.
  • labeled dideoxynucleotide and DNA polymerase are added to detect the presence or absence of an extension reaction.
  • Various kinds of labels such as fluorescent dyes, radioactive substances, and compounds that can be detected electrochemically can be selected.
  • the specific hybridization means specific hybridization under normal hybridization conditions, preferably under highly stringent hybridization conditions.
  • highly stringent conditions refer to, for example, hybridization in a 65 ° C. solution of at least about 6 ⁇ SSC and 1% SDS, and an initial wash at 20 ° C. of 20% (v / v) formamide ( (In 0.1 ⁇ SSC) for 10 minutes, and the following washing can be performed under conditions of 0.2 ⁇ SSC and 0.1% SDS at 65 ° C., but is not limited thereto, and those skilled in the art can appropriately select the conditions. .
  • Other methods for detecting the polymorphism at position -184 include using a mass spectrometer (MALDI-TOF-MS, etc.) and detecting the polymorphism based on the difference in mass; alleles labeled with a quencher and a fluorescent dye.
  • MALDI-TOF-MS mass spectrometer
  • TaqMan PCR method in which PCR is performed using specific oligo and Taq DNA polymerase; so-called invader method; rolling circle method; method for analyzing sample DNA sequence using sequencer, denatured HPLC method, Melting TM temperature
  • invader method so-called invader method
  • rolling circle method method for analyzing sample DNA sequence using sequencer, denatured HPLC method, Melting TM temperature
  • methods include, but are not limited to, analysis, PCR-SSOP (sequence-specific oligo nucleotide probe) method, PCR-PHFA (preferential homo-formation assay) method, PCR-RSCA (reference strand conformation assay) method, and the like.
  • the second embodiment of the method for examining coronary artery disease risk in the present invention includes a step of detecting an increase in MKL1 gene expression in a sample collected from a subject.
  • the method for detecting the enhancement of MKL1 gene expression is not particularly limited, and a gene expression analysis method known to those skilled in the art can be used. For example, it can be detected at the transcription level or the translation level, or it can be detected by measuring the function or the like of the promoter, but is not limited thereto. Hereinafter, this method will be specifically described.
  • the present inventors have found that the expression of MKL1 gene is significantly enhanced at the protein level in human coronary atherosclerotic site pathology specimens obtained from patients with severe coronary stenosis . By immunostaining, MKL protein was strongly expressed in foamed macrophages, and expression of MKL1 protein was also confirmed in ⁇ -actin positive smooth muscle cells.
  • the present inventors have determined by a reporter gene assay that the polymorphism at position -184 is involved in the expression of the MKL1 gene at the transcription level, and further has a polymorphism of -184C> T.
  • gel shift assay confirms that the affinity between a nuclear factor that is considered to be a transcriptional repressor and the MKL1 gene near position -184 is reduced, and the mechanism of the enhancement of MKL1 gene expression by -184C> T is confirmed. Revealed.
  • Atherosclerosis is thought to be an inflammatory response of macrophages and lymphocytes to pathogenic lipoproteins that erode arterial vascular walls (Libby, P. et al., Circulation., 105, 2002, 1135-1143; Glass , C, K. et al., Cell., 104, 2001, 503-516).
  • the MKL1 gene is known as a transcriptional cofactor and may play an important role in the regulation of cholesterol uptake into cells and the expression of cytokines in macrophages during atherogenesis.
  • Activated macrophages are obtained from a subject by blood collection, surgery, or intimal biopsy.
  • the method for measuring the expression of the MKL1 gene in activated or foamed macrophages is not particularly limited, and a method known to those skilled in the art or a method equivalent thereto can be used, and it can be detected at either the transcription level or the translation level. May be.
  • MKL1 gene expression at the transcription level can be performed by a method known to those skilled in the art or a method analogous thereto, and examples thereof include the following methods.
  • RNA is extracted according to a conventional method, and this is heated in a solution containing glyoxal, formamide, formalin, methylmercury, etc., and intramolecular hydrogen bonding is performed. Detach and break the higher order structure into a line. Thereafter, electrophoresis is performed on an agarose gel containing formalin. Transfer the gel to a nylon membrane or nitrocellulose membrane with a high salt solution of 15-20 x SSC. In the case of a nitrocellulose membrane, RNA is fixed by treating in a vacuum oven at 80 ° C for about 2 hours.
  • the RNA is immobilized by crosslinking by irradiating with ultraviolet rays.
  • a probe is prepared from the cloned cDNA, labeled, and after contacting the probe and membrane under specific conditions, only the mRNA bound to the cDNA probe is detected. it can.
  • Hybridization conditions can be appropriately selected by those skilled in the art depending on salt concentration, temperature, base length, composition, and the like.
  • RNA sample is subjected to a reverse transcription reaction using a reverse transcriptase and an oligo (dT) primer, the obtained cDNA is amplified by a PCR method, and then the cDNA is detected using a labeled complementary nucleic acid.
  • Dot blot method A modification of the Northern blot method, in which mRNA extracted from activated macrophages derived from a subject is denatured by the methylmercury hydroxide method, etc. Spotting on the filter as dots (dots) and hybridization with a probe labeled with a radioactive label or the like detects the intensity of the signal. Although it is simpler than the Northern blot method, there is a possibility that another mRNA having a homology to the probe or one having a different length from the target mRNA may be detected.
  • RNase protection assay This is a method that utilizes the property that RNase does not degrade double-stranded RNA hybridized in perfect agreement with the target RNA, but cleaves when there is a mismatch.
  • mRNA labeled with 32 P or the like is used as a probe to hybridize with sample RNA, and then digested with RNAse.
  • the reaction product is electrophoresed on an agarose gel or polyacrylamide gel, and the size is measured. If the transcript is completely complementary to the probe RNA, a large single band is displayed, but if there is a mismatch, the size of the band will be reduced or two or more bands will be generated. The presence or absence of RNA can be confirmed.
  • detection of MKL1 gene expression at the transcription level can also be performed by a method using a DNA microarray.
  • detection of MKL1 gene expression at the translation level can be carried out by a method known to those skilled in the art or a method analogous thereto, and examples thereof include the following methods.
  • Immunostaining This is a method of chemically fixing a protein in a tissue sample and detecting its localization. For example, it can be performed by the following procedure. A tissue containing activated macrophages derived from a subject is washed with PBS or the like, and then a frozen section of about 5 to 20 ⁇ m is prepared according to a conventional method and attached to a silane-coated slide glass or the like. The sections are air-dried and then fixed in paraformaldehyde solution on ice for 10-30 minutes.
  • the test agent for coronary artery disease risk is a nucleic acid that hybridizes to a nucleic acid consisting of a region of at least 10 bases including position 817 in the base sequence set forth in SEQ ID NO: 1, or the sequence set forth in SEQ ID NO: 1. It includes a nucleic acid that hybridizes to a nucleic acid consisting of a region complementary to a nucleic acid consisting of a region consisting of at least 10 bases including position 817 in the base sequence.
  • nucleic acid can be used as a probe for detecting a target DNA or RNA sequence in the above-described method for detecting the polymorphism at position -184 or the method for detecting MKL1 gene expression at the transcription level.
  • the nucleic acid can be synthesized by a method known to those skilled in the art. At the time of synthesis, a method using a nucleotide derivative labeled with a fluorescent dye or the like, or a method of labeling the synthesized nucleic acid with a fluorescent dye or the like Can also be detectably modified.
  • the nucleic acid is preferably 10 to 100 bases long, more preferably 15 to 50 bases long, and further preferably 15 to 30 bases long.
  • a second aspect of the coronary artery disease risk test agent according to the present invention is a nucleic acid consisting of a nucleic acid consisting of a region of at least 10 bases including position 817 in the base sequence shown in SEQ ID NO: 1, or a nucleic acid consisting of a region complementary thereto.
  • Including primers capable of amplifying The primer is used in the above-described method for detecting the polymorphism at position -184 and the method for detecting MKL1 gene expression at the transcription level. There is no particular limitation as long as the region including the -184 position can be amplified, and the length of the amplified region is also arbitrary.
  • the primer can be appropriately designed and synthesized by a method known to those skilled in the art based on the base sequence of the region to be amplified.
  • the primer is preferably 10 to 100 bases long, more preferably 15 to 50 bases long, and still more preferably 15 to 30 bases long.
  • the third aspect of the test agent for coronary artery disease risk according to the present invention includes an antibody against MKL1 protein.
  • Such an antibody is used, for example, in detecting MKL1 protein in the above-described method for detecting MKL1 gene expression at the translation level.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • Monoclonal antibodies can be produced by isolating antibody-producing cells from animals immunized with MKL1 protein, fusing them with myeloma cells and the like to produce hybridomas, and producing the hybridomas.
  • Polyclonal antibodies can be obtained from the sera of animals immunized with MKL1 protein.
  • the antibody contained in the test drug according to the present invention can be labeled with a fluorescent substance, a radioactive substance or the like.
  • the test agent may contain a secondary antibody for detecting an anti-MKL1 protein antibody.
  • test drug according to the present invention may contain various enzymes, enzyme substrates, buffer solutions, and the like depending on the test method.
  • the present invention also includes a method for diagnosing coronary artery disease risk, which comprises the step of detecting the presence or absence of a mutation that enhances MKL1 gene expression or the presence or absence of enhancement of MKL1 gene expression.
  • the step of detecting the presence or absence of a mutation that enhances MKL1 gene expression or the presence or absence of enhancement of MKL1 gene expression can be performed in the same manner as in the test method according to the present invention, and thus the description thereof is omitted here.
  • MKL1 gene is enhanced in activated macrophages accumulated in lipids in atherosclerotic lesions and smooth muscle cells in neointimal by immunohistochemical staining did. This strongly suggests that the MKL1 gene is one of the causes of coronary artery disease.
  • the method for preventing or treating coronary artery disease according to the present invention includes a step of inhibiting or suppressing the expression of the MKL1 gene or the function of the MKL1 protein. Inhibition or suppression of gene expression can be performed according to methods known to those skilled in the art, and examples thereof include the following methods.
  • Antisense method RNA having a sequence complementary to the transcription product of the MKL1 gene (antisense RNA) is introduced into cells to suppress the function of the transcription product.
  • the sequence of the antisense RNA can be designed based on the base sequence of the target MKL1 gene.
  • chemical modification may be performed according to a known method.
  • Antisense RNA can be obtained by inserting a DNA encoding the antisense RNA into an expression vector, introducing it into a cell and transcribing it inside the cell, or introducing an antisense RNA synthesized outside the cell into the cell. Introduced in.
  • RNA molecules synthesized outside the cell When producing by transcription in a cell, it can be efficiently transferred into the cytoplasm by providing a signal sequence.
  • a lipofection method embedded in liposomes or the like when administering an RNA molecule synthesized outside the cell, a lipofection method embedded in liposomes or the like may be used. By adding an antibody to the liposome surface, it can be efficiently transferred to the target cells.
  • Ribozyme method This method utilizes RNA molecules (ribozymes) that hydrolyze and cleave a predetermined position of RNA.
  • a ribozyme consists of a positioning sequence for recognizing a cleavage site and a catalytic sequence having catalytic activity. By making the positioning sequence complementary to a predetermined position of the transcription product of the MKL1 gene, the transcription product is cleaved and the expression of the MKL1 gene is suppressed.
  • the base sequence of ribozyme is designed according to a known method.
  • ribozymes can be inserted into an expression vector by inserting a ribozyme-encoding DNA into the cell and transcribed inside the cell, or antisense RNA synthesized outside the cell can be introduced into the cell. Is introduced into the cell.
  • antisense RNA it is also preferable to perform chemical modification and addition of a signal sequence.
  • RNAi method In this method, the function of the target gene is specifically disrupted by double stranded RNA (dsRNA).
  • dsRNA double stranded RNA
  • MKL1 gene cDNA as a template and introducing it into cells, or by expressing hairpin RNA and expressing double-stranded RNA in cells.
  • the base sequence of dsRNA is designed based on the base sequence of MKL1 gene.
  • RNA or DNA molecules capable of binding to a target substance.
  • aptamers specifically bind to MKL1 protein, thereby suppressing the physiological activity of MKL1 protein in cells.
  • Aptamers that target MKL1 protein or nucleic acid encoding MKL1 protein can be selected, for example, from a randomly synthesized DNA library according to a known screening method.
  • Decoy method uses a short nucleic acid (decoy) containing the sequence of the transcription factor binding site of the target gene.
  • the decoy is a decoy for the transcription factor and inhibits the transcription factor from binding to the transcription factor binding site of the target gene.
  • the decoy can be designed based on the base sequence of the transcription factor binding site of the MKL1 gene.
  • Trans-splicing method This is a method of repairing mutant mRNA by a ribozyme (trans-splicing ribozyme) to which a nucleic acid having a normal base sequence is added.
  • a trans-splicing ribozyme can be designed to cut off the region containing the -184 position in MKL1 mRNA, and by adding a nucleic acid with cytosine at -184 position to the trans-splicing ribozyme.
  • the nucleic acid can be linked to MKL1 mRNA.
  • An antibody that inactivates the MKL1 protein may be administered to inhibit the function of the MKL1 protein.
  • An antibody against the MKL1 protein can be prepared by the same method as described for the above-mentioned test agent, and screening can be performed to select those that inactivate the MKL1 protein.
  • the method for screening a prophylactic or therapeutic agent for coronary artery disease comprises a step of contacting a cell capable of expressing the MKL1 gene with a prophylactic or therapeutic drug candidate, and the therapeutic agent candidate that suppresses the expression of the MKL1 gene.
  • Selecting Cells capable of expressing the MKL1 gene can be prepared according to methods known to those skilled in the art. For example, various host cells are transformed with a vector containing the MKL1 gene having a polymorphism of -184C> T in the promoter region. Can be obtained. Host cells and vectors can be appropriately selected and used by those skilled in the art.
  • a candidate for a prophylactic or therapeutic agent for coronary artery disease is administered to the cells expressing the MKL1 gene thus obtained to detect whether the expression is suppressed.
  • Prophylactic or therapeutic drug candidates are not particularly limited, and examples thereof include pharmaceuticals containing low molecular weight compounds, proteins, and nucleic acids.
  • the nucleic acid include the above-described antisense RNA, ribozyme, dsRNA, aptamer, decoy and the like, and examples of the protein include antibodies.
  • RNA and protein can be administered as they are, or can be administered in a form that is transcribed and translated in cells.
  • MKL1 gene expression can be performed using the method described above.
  • the present invention also includes DNA encoding the MKL1 protein, which consists of a base sequence in which the base corresponding to position 817 in the base sequence set forth in SEQ ID NO: 1 is T.
  • DNA is a novel gene sequence encoding MKL1 protein containing a novel single nucleotide polymorphism of ⁇ 184C> T in the promoter region.
  • the DNA has a lower affinity for a nuclear factor that suppresses expression than the known MKL1 protein gene, and as a result, the MKL1 protein is highly expressed.
  • the present invention also includes a recombinant vector containing such DNA and a transformant containing the recombinant vector.
  • the recombinant vector and transformant can be prepared by those skilled in the art according to a known method by selecting an optimal vector and host, and have a remarkable effect of highly expressing the MKL1 protein. is there.
  • [Target] A total of two groups of 1,738 patients with coronary artery disease and 3,354 controls were studied.
  • the first group Japanese group A
  • Screening of coronary artery disease-related gene loci using genome-wide microsatellite markers was performed using 392 case samples arbitrarily selected from here and 392 control samples.
  • Japanese group B Japanese group B is the Gifu Prefectural General Medical Center, Gifu Prefectural Tarimi Hospital, Hirosaki University Hospital, Reimei Rehabilitation Hospital, and Hirosaki Stroke Center, who visited the outpatients with various symptoms, or for health examinations. 3,231 (including 1,109 patients with myocardial infarction and 2,122 controls). Table 1 shows the properties of each sample.
  • HT hypertension, hypertension 140 mmHg or higher, minimum blood pressure 90 mmHg or higher, or antihypertensive agent
  • HC hyperlipidemia Mean that serum total cholesterol is 5.72 mmol / l (220 mg / dl) or who is taking a lipid-lowering drug
  • DM indicates diabetes, fasting blood glucose is 6.93 mmol / l (126 mg / dl), glycosyl Those who have a hemoglobin content of 6.5% or more, or who are taking antidiabetic drugs.
  • Coronary atherosclerosis was classified as 0, 1, 2 or 3 vessel disease (VD) according to the number of coronary arteries with severe stenosis (luminal stenosis by angiography> 50%).
  • VD vessel disease
  • Controls include individuals with no history of coronary heart disease, peripheral arterial occlusive disease or other atherosclerosis; ischemic or hemorrhagic stroke or other brain disease; thrombotic, embolic or bleeding disorders did. Each disease was diagnosed according to normal diagnostic criteria. Informed consent was given to each participant, and this study was approved by the ethics committee of each medical institution.
  • a set of first to third DNA sample pools was stocked according to the method of Tamiya et al. (Tamiya, G. et al. Hum Mol Genet., 2005, 2305-2321). Specifically, the first set is 100 cases of 3VD myocardial infarction, 100 controls, the second set is 100 cases of 2VD myocardial infarction, 100 controls, the third set is 50 years of onset There were 192 myocardial infarction cases (2 cases of 3VD, 46 cases of 2VD, 144 cases of 1VD) and 192 controls.
  • DNA sample stock preparation use double-stranded DNA quantification kit (Pico Green from MolecularMProbes) according to the method of Collins et al. (Collins, HE et al., Hum Genet. 106, 2000, 218-226) Then, the genomic DNA concentration was measured in triplicate, and each genomic DNA was adjusted to 8 ng / ⁇ l.
  • MS markers associated with coronary artery disease were identified by a case control study using the first case / control DNA sample pool set.
  • the second case / control for MS markers that showed a significant association with coronary artery disease in the first screening (p ⁇ 0.05 in 2 by 2 analysis and / or 2 by multiple analysis) Analysis using a set of DNA sample pools. That is, the allele frequency in each DNA sample pool was determined by the peak height, and each allele frequency was obtained by dividing the peak height of each allele by the sum of the peak heights of all alleles. . Differences in allelic frequencies between the two DNA samples were evaluated by chi-square test using 2 ⁇ 2 and 2 ⁇ m (m is the number of alleles) contingency tables. Six MS markers were evaluated by Fisher's exact test. In all experiments, a P value of 0.05 or less was considered significant.
  • MS markers that yielded statistically significant results in both the first and second screening For MS markers that yielded statistically significant results in both the first and second screening, the individual samples that make up the first and second DNA sample sets are typed (individual typing) The relevance of was verified. MS markers that have been found to be significantly associated with coronary artery disease are individually identified using a third case and control DNA sample set to test the hypothesis that these MS markers are associated with coronary artery disease. It was subjected to a third screening by typing. Thus, by performing a multi-step screening using a plurality of DNA sample sets, it is possible to reduce the possibility of false positives relating to a disease.
  • FIG. 1 The outline of the genome-wide screening described above is shown in FIG. 1, and the results are shown in FIG. In the first screening, 1,818 MS markers were significantly associated with coronary artery disease, of which 190 MS markers remained after the second screening. When these MS markers were individually typed for 96 cases and 96 controls arbitrarily extracted from the first and second sets, 42 MS markers showed a significant association. Furthermore, similar results were obtained by screening using another 96 cases and 96 controls arbitrarily extracted from the first and second sets.
  • MS markers were identified as 6 MS markers related to coronary artery disease.
  • the six markers are shown in Table 2.
  • the linkage disequilibrium (LD) structure around these six MS markers was analyzed based on HapMap JPT + CHB data.
  • D ′ and r 2 were calculated with Haploview software.
  • CAD3, CAD4, and CAD5 are located in LD blocks that do not contain known genes
  • CAD1, CAD2, and CAD6 exist in LD blocks that contain known genes MKL1, PCSK9, and K125001, respectively.
  • genotyping of the PCSK9 gene revealed that CAD2-MS showed a specific association with hypercholesterolemia. The results are shown in Table 3.
  • CAD1-MS located in the MKL1 gene is present on chromosome 22q13 (FIG. 3).
  • the recombination hotspot predicted from the HapMap data (Phase II, release 21) was found to be 10 kb upstream and 3 ′ untranslated region of MKL1, and only the MKL1 gene was contained in the CAD1-related LD block.
  • CAD1-MS was further genotyped for cases and controls. The genotype distribution and SNP allele frequency were compared by chi-square test, and the strength of association was expressed by odds ratio (OR). The results are shown in Table 4.
  • SNP analysis in MKL1 Furthermore, SNP analysis was performed on the locus related to coronary artery disease. The relationship between specific SNPs and diseases was further verified by replication studies using different groups (Japanese group B).
  • Reporter assays were performed using a luciferase reporter construct containing the -184C> T allele. Based on the transcription start site database, a 249 bp fragment of the promoter region of the MKL1 gene was determined and amplified by PCR using the following primer pairs. 5'-CCACGCGTTCCTCAGAGATGAGGTCAT-3 '(SEQ ID NO: 14) 5'-TGCTCGAGCCGGCTCCTCTCAGCCACGG-3 '(SEQ ID NO: 15)
  • Genomic DNA was obtained from homozygous individuals with -184C> T and used as a template.
  • the PCR product was digested with MluI and XhoI restriction enzymes and inserted upstream of the firefly luciferase gene of pGL3-basic vector (Promega).
  • K562 or HeLa cells were cultured in RPMI medium-1840 or Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin 1% under conditions of 37 ° C. and 5% CO 2 .
  • Each construct was introduced into K562 cells (2 ⁇ g each) by electroporation or HeLa cells (1 ⁇ g each) by lipofection together with 0.05 ⁇ g of pRL-tk (Promega) as an internal control.
  • Cells were collected 24 hours after transfection, and a cell lysate was prepared according to the method of Shibata et al. (Shibata, H. et al. Hum Immunol, 67, 2006, 363-373).
  • the luciferase activity of each cell lysate was determined using the DUAL Luciferase Reporter Assay System (Promega) and firefly luciferase levels were normalized to the Renilla luciferase levels from pRL-tk. Data were collected in 4 independent experiments (each quadruplicate).
  • the luciferase activity driven by the sensitive allele T is 1.2 times higher than the transcription activity of the resistant allele C in both K562 cells (FIG. 4A) and HeLa cells (FIG. 4B).
  • FIG. 4A the luciferase activity driven by the sensitive allele T is 1.2 times higher than the transcription activity of the resistant allele C in both K562 cells (FIG. 4A) and HeLa cells (FIG. 4B).
  • the selected cell line was RPMI medium-1840 supplemented with 10% FBS and 1% penicillin / streptomycin, cultured at 5% CO 2 , collected at the growth stage, and then used with RNeasy mini kit (Qiagen) Total RNA was obtained from about 10 7 cells.
  • Total RNA (5 ⁇ g) was subjected to cDNA synthesis by Superscript II reverse transcriptase (Invitrogen).
  • Quantitative real-time PCR was performed using iCycler iQ Real-Time PCR Detection System (Bio-Rad) and SYBR Premix Ex Taq (Takara) to measure the relative amount of mRNA.
  • the relative amount of transcript of MKL1 gene was normalized by dividing by the relative amount of GAPDH.
  • the primer pairs used are as follows.
  • MKL1 5'-TAGCCGATGACCTCAATGAG-3 '(SEQ ID NO: 16) 5'-ATCGAAGGAAGAGCTGTCTG-3 '(SEQ ID NO: 17)
  • GAPDH 5'-CTTCACCACCATGGAGAAGGC-3 '(SEQ ID NO: 18) 5'-GGCATGGACTGTGGTCATGAG-3 '(SEQ ID NO: 19)
  • the experiment was repeated three times, and in each experiment the sample was triplicated.
  • the B cell line having the T allele homozygote had about 1.8 times (1.5 to 2.1 times) higher MKL1 gene expression than the C allele homozygote. This further supports that the -184T allele is involved in the increased expression of the MKL1 gene.
  • DIG digoxigenin
  • Nuclear extracts were prepared from K562 cells using Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific (Rockford)). The DIG-labeled probe and K562 nuclear extract were incubated at 4 ° C. for 30 minutes and then separated by electrophoresis on a 6% non-denaturing polyacrylamide gel with 0.5 ⁇ TBE running buffer.
  • the DNA-protein complex was electroblotted onto a positively charged nylon membrane and visualized according to the manual for DIG-Gel-Shift-Kit-2nd generation.
  • K562 nuclear extracts were pre-incubated with various concentrations of unlabeled competitor for use in competition assays.
  • the DNA-protein complex showed higher affinity at -184C than at -184T.
  • this DNA-protein interaction was more competitive with unlabeled DNA when the -184C probe was included than when the -184T probe was included (FIG. 5B).
  • TFSEARCH http://mbs.cbrc.jp/research/db/TFSEARCHJ.html
  • EMSA band supershift large electrophoretic delay
  • Atherosclerotic lesions were collected by pathoanatomy of patients with severe coronary stenosis and immunohistochemically stained.
  • tissue section embedded in paraffin was deparaffinized with xylene and hydrated with deionized water from ethanol.
  • Tissue sections were blocked with DakoCytomation Protein Block (DAKO) for 5 minutes, and incubated with anti-human MKL1 polyclonal antibody (Santa Cruz; sc-32909) at 1: 100 dilution for 2 hours at room temperature. After washing with a washing buffer and a phosphate buffer solution, incubation with a 3% aqueous hydrogen peroxide solution for 5 minutes blocked the endogenous peroxidase activity.
  • DAKO DakoCytomation Protein Block
  • the sections were washed with a washing buffer and a phosphate buffer solution, then incubated with a secondary antibody (DAKO ChemMate Envision / HRP), followed by incubation with a DAB chromogen, and counterstaining with Mayer's hematoxylin.
  • DAKO ChemMate Envision / HRP secondary antibody
  • Immunohistochemical analysis of adjacent tissues using cell type specific antibodies was performed using anti-human CD68 monoclonal antibody (DAKO) and anti-human smooth muscle ⁇ -actin monoclonal antibody (DAKO).
  • MKL1 protein is expressed in the lipid core of atherosclerotic plaque.
  • the adjacent region was stained with an anti-CD68 antibody, it was found that the MKL1-positive cells were foamed macrophages (see FIG. 6B).
  • MKL1 protein was also expressed in smooth muscle ⁇ -actin positive cells in the neointima (FIG. 6C).
  • SEQ ID NO: 1 is the base sequence of the MKL1 genomic gene containing an upstream sequence 1000Bp and a downstream sequence 500bp.
  • SEQ ID NO: 2 is a forward primer for amplifying the MS marker CAD1-MS.
  • SEQ ID NO: 3 is a reverse primer for amplifying the MS marker CAD1-MS.
  • SEQ ID NO: 4 is a forward primer for amplifying the MS marker CAD2-MS.
  • SEQ ID NO: 5 is a reverse primer for amplifying MS marker CAD2-MS.
  • SEQ ID NO: 6 is a forward primer for amplifying the MS marker CAD3-MS.
  • SEQ ID NO: 7 is a reverse primer for amplifying MS marker CAD3-MS.
  • SEQ ID NO: 8 is a forward primer for amplifying MS marker CAD4-MS.
  • SEQ ID NO: 9 is a reverse primer for amplifying the MS marker CAD4-MS.
  • SEQ ID NO: 10 is a forward primer for amplifying MS marker CAD5-MS.
  • SEQ ID NO: 11 is a reverse primer for amplifying MS marker CAD5-MS.
  • SEQ ID NO: 12 is a forward primer for amplifying the MS marker CAD6-MS.
  • SEQ ID NO: 13 is a reverse primer for amplifying the MS marker CAD6-MS.
  • SEQ ID NO: 14 is a forward primer for amplifying a fragment of the promoter region of MKL1 gene.
  • SEQ ID NO: 15 is a reverse primer for amplifying a fragment of the promoter region of MKL1 gene.
  • SEQ ID NO: 16 is a forward primer used for quantitative real-time PCR on MKL1 transcript in B cell line.
  • SEQ ID NO: 17 is a reverse primer used for quantitative real-time PCR on MKL1 transcript in B cell line.
  • SEQ ID NO: 18 is a forward primer used for quantitative real-time PCR for GAPDH transcripts in B cell lines.
  • SEQ ID NO: 19 is a reverse primer used for quantitative real-time PCR on GADPH transcript in B cell line.
  • SEQ ID NO: 20 is a forward primer for -184C allele used for EMSA.
  • SEQ ID NO: 21 is a reverse primer for the -184C allele used for EMSA.
  • SEQ ID NO: 22 is a forward primer for -184T allele used for EMSA.
  • SEQ ID NO: 23 is a reverse primer for the -184T allele used for EMSA.

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Abstract

La présente invention concerne : une méthode de détermination des maladies coronariennes utilisant un SNP dont la corrélation aux pathologies a été entièrement démontrée, etc.; un réactif utilisable dans la méthode; etc. La présente invention concerne spécifiquement une méthode de détermination du risque de maladies coronariennes chez un patient, ladite méthode comprenant une étape de détection d'une mutation qui provoque l'augmentation du niveau d'expression du gène MKL1 ou la fréquence augmentation du niveau d'expression d'un gène MKL1 dans un échantillon récolté chez le sujet.
PCT/JP2009/006918 2008-12-24 2009-12-16 Méthode de détermination du risque de maladies coronariennes WO2010073544A1 (fr)

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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DATABASE DDBJ/EMBL/GENBANK [online] 7 September 2001 (2001-09-07), retrieved from http://www.ncbi.nlm.nih.gov/ sviewer/viewer.fcgi?14161366:NCBI:2651775 Database accession no. AF368061 *
DATABASE POST GENOME JIDAI NO IDEN YODOSHA CO., LTD.; 10 March 2002 (2002-03-10), NAOYUKI KAMATANI ET AL. *
HINOHARA, K. ET AL.: "Megakaryoblastic leukemia factor-1 gene in the susceptibility to coronary artery disease", HUM. GENET., vol. 126, no. 4, October 2009 (2009-10-01), pages 539 - 547 *
KAWASHIMA, M. ET AL.: "Genomewide association analysis of human narcolepsy and a new resistance gene", AM. J. HUM. GENET., vol. 79, no. 2, August 2006 (2006-08-01), pages 252 - 263 *
MA, Z. ET AL., DEFINITION: HOMO SAPIENS MEGAKARYOBLASTIC LEUKEMIA-1 PROTEIN(MKL1) MRNA, 9 February 2010 (2010-02-09) *
MA, Z. ET AL.: "Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia", NAT.GENET., vol. 28, no. 3, July 2001 (2001-07-01), pages 220 - 221 *
MITSUHIRO YOKOTA ET AL.: "Shikkan Genome Kaiseki 4. Idenshitakei ni yoru Kandomyaku Shikkan Risk no Yosoku", EXPERIMENTAL MEDICINE, vol. 23, no. 4, 15 February 2005 (2005-02-15), pages 49 - 58 *
YASUHARU TABARA ET AL.: "Idenshi Kaiseki no Shinpo Genome Wide Sokan Kaiseki ni yoru Koketsuatsu Kanjusei Idenshi no Tansaku - Millennium Genome Project", MOLECULAR MEDICINE, vol. 42, 5 September 2005 (2005-09-05), pages 208 - 213 *
YOSHIJI YAMADA ET AL.: "Bunshi Level no Saishin Shikkan Kenkyu 11) Idenshitakei ni yoru Shinkin Kosoku Kikendo no Yosoku", EXPERIMENTAL MEDICINE, vol. 21, no. 9, 1 June 2003 (2003-06-01), pages 1258 - 1262 *

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