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WO2006117945A1 - Technique de detection de troubles du metabolisme des lipides et agent diagnostique utilise avec ces technique - Google Patents

Technique de detection de troubles du metabolisme des lipides et agent diagnostique utilise avec ces technique Download PDF

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WO2006117945A1
WO2006117945A1 PCT/JP2006/305909 JP2006305909W WO2006117945A1 WO 2006117945 A1 WO2006117945 A1 WO 2006117945A1 JP 2006305909 W JP2006305909 W JP 2006305909W WO 2006117945 A1 WO2006117945 A1 WO 2006117945A1
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fatty acid
fabpl
lipid metabolism
metabolism disorder
base
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Toshiji Saibara
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Kyushu University NUC
Kochi University NUC
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Kyushu University NUC
Kochi University NUC
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the present invention relates to a method for detecting and judging lipid metabolism disorders and a risk of developing diseases caused thereby (hereinafter such a disease is sometimes referred to as a "lipid metabolism disorder-related disease”) . More specifically, the method of the present invention detects, from a sample containing human genomic DNA, an SNP (Single Nucleotide Polymorphism) associated with lipid metabolism disorders, or a variation in an amino acid sequence due to the polymorphism, to thereby detect and judge whether the subject has a lipid metabolism disorder, or whether the subject has a potential risk of developing a lipid metabolism-related disease.
  • SNP Single Nucleotide Polymorphism
  • the present invention relates to reagents, such as a probe and primer, for lipid metabolism disorder diagnosis, and a reagent kit comprising at least one of such reagents, all of which can be effectively used in the detection method.
  • the present invention also relates to a method for screening for components that are effective for alleviating lipid metabolism disorders or lipid metabolism disorder-related diseases or decreasing a risk of developing the said disease.
  • the present invention further provides a method for evaluating therapeutic effects of a medicine or other treatment on lipid metabolism disorders or lipid metabolism disorder- related diseases caused by genetic polymorphisms.
  • Metabolic syndrome is a morbid condition in which hypertension, hyperlipemias (hypertriglyceridemias, hypo-HDL cholesterolemia) , visceral lipid storage diseases, carbohydrate metabolism abnormality, insulin resistance, etc. are clustered because of metabolic balance breakdown due to obesity, with the result that arteriosclerotic diseases are likely to develop.
  • the syndrome is attracting attention due to the recent increase of patients with lifestyle- related diseases, such as obesity and fatty liver.
  • metabolic syndrome There are various risk factors associated with metabolic syndrome. In addition to genetic factors such as MODY genes and thrifty genes, environmental factors such as obesity are implicated in the onset of the syndrome.
  • researchers are gradually identifying some candidate genes and chromosomal regions that specify susceptibility to metabolic syndrome, mainly from the results of genome wide linkage analyses. Such identification of genetic predispositions to metabolic syndrome is important for accurate diagnosis of the syndrome and effective clinical intervention based on the diagnosis, and is expected to make a significant contribution to preventive medicine in particular.
  • NASH non-alcoholic steatohepatitis
  • liver disease in which, although the patient has no alcohol drinking habit, tissue is observed which is remarkably similar to that observed in alcoholic hepatitis having fatty liver as a background lesion, and NASH is very frequently accompanies visceral obesity and insulin resistance.
  • NASH is known to progress to serious diseases, such as liver cirrhosis and liver cancer, and to prevent the progression of NASH to such potential liver cirrhosis, it is necessary to find NASH at an early stage and perform medical treatment.
  • SNPs are high-density DNA polymorphisms, and occur at a frequency of one per about 300 base pairs. Thus, it is highly likely that an SNP exists in the vicinity of any given gene. Recent technology is making it possible to identify disease-causing genes from a region narrowed by a linkage analysis, by using an SNP as a genetic disease marker.
  • the genetic usefulness of SNPs as genetic markers is especially noticeable in polygenetic diseases in which a great number of genes are involved in a complex manner. When SNPs themselves cause qualitative and/or quantitative changes in genetically expressed products, they can be direct diagnostic or therapeutic targets.
  • Metabolic syndrome including lifestyle-related diseases is viewed as a polygenetic syndrome associated with a large number of genes, but the basis of the syndrome is metabolic balance breakdown due to obesity.
  • Non-Patent Document 1 Saibara T, et al., 'Non-alcoholic steatohepatitis' , Lancet. 354: 1299-1300, 1999
  • Non-Patent Document 2 Yamamura N, et al., "Pharmacokinetic analysis of 123 I-labeled medium chain fatty acid as a radiopharmaceutical for hepatic function based on beta-oxidation' , Ann Nucl Med. 13:235-239, 1999
  • Non-Patent Document 3 Brouillette C, et al., ⁇ ffect of liver fatty acid binding protein (FABP) T94A missense mutation on plasma lipoprotein responsiveness to treatment with fenofibrate' , J Hum Genet, 49: 424-432, 2004
  • Non-Patent Document 4 Lawrence Chan, et al., "Human Liver Fatty Acid Binding Protein cDNA and Amino Acid Sequence' , THE Journal of Biological Chemistry, Vol.260, No.5, pp.2629-2632, 1985
  • An object of the present invention is to identify a genetic polymorphism associated with lipid metabolism disorders, and a lipid metabolism disorder susceptibility gene (i.e., a genetic predisposition to lipid metabolism disorders) .
  • a further object of the present invention is to provide a method for detecting or judging, based on the identification, a lipid metabolism disorder and a risk of developing a disease caused thereby.
  • a still further object of the present invention is to provide a reagent and reagent kit that are useful for conveniently performing the method.
  • Another object of the present invention is to provide a method for searching for an active ingredient for a preventive or alleviating agent for a lipid metabolism disorder or a disease caused thereby (lipid metabolism disorder-related disease) , based on the finding about the genetic polymorphism associated with lipid metabolism disorders, and the lipid metabolism disorder susceptibility gene.
  • Yet another object of the present invention is to provide a method for evaluating a therapeutic effect of a treatment on a lipid metabolism disorder or disease caused thereby (lipid metabolism disorder-related disease) , or disease risk-reducing effect of a treatment.
  • the present inventors conducted extensive research for the purpose of finding a gene associated with a lipid metabolism disorder that can be a factor of metabolic syndrome. As a result, the inventors found that a group of subjects with insufficient hepatic fatty acid beta-oxidation ability have a homozygotic genetic polymorphism (genetic abnormality) which is clearly different from that of normal persons, in the base sequence of gene of the hepatic fatty acid binding protein (FABPl: fatty acid binding protein 1, liver) at locus 2pll on human chromosome 2, and succeeded in identifying the polymorphism.
  • FABPl hepatic fatty acid binding protein 1, liver
  • NASH non-alcoholic steatohepatitis
  • NASH patients can be divided into two types: those in whom fatty acids are metabolized in the liver in a normal manner (normal hepatic fatty acid metabolism-type) , and those in whom fatty acids are not normally metabolized in the liver (hepatic fatty acid metabolism disorder-type) , and that humans of hepatic fatty acid metabolism disorder-type are genetically (inherently) unable to compensatorily enhance fatty acid metabolism in the liver, and have a high risk of developing NASH.
  • this genetic polymorphism occurs with high frequency, not only in patients with NASH of hepatic fatty acid metabolism disorder-type, but also in patients with myocardial infarction, and that the genetic polymorphism (genetic abnormality) serves as a rate limiting step of hepatic fatty acid beta-oxidation, and is a genetic predisposition to fatty acid and neutral fat metabolism disorders not only in the liver but also in the entire body.
  • the inventors are sure that the genetic polymorphism (genetic abnormality) can be advantageously used as a diagnostic and therapeutic target, since it is located in an exon of the FABPl gene and changes an amino acid of FABPl to thereby cause qualitative and/or quantitative change in FABPl.
  • the present inventors are sure that, in individual patients, a lipid metabolism disorder or a potential risk of developing a disease caused by the disorder can be accurately detected by detecting a genetic polymorphism (genetic abnormality) in the FABPl gene, or a qualitative or quantitative change in FABPl due to such a polymorphism.
  • the present invention was thus accomplished.
  • the present invention encompasses the following aspects.
  • Method for detecting lipid metabolism disorder or risk of disease caused thereby and a reagent for use in the method (1-1) •
  • a method for detecting a lipid metabolism disorder or a risk of a disease caused thereby comprising the step of detecting, in a biological sample from a subject, a genetic polymorphism that causes change (T-»A) of the 94th amino acid of FABPl [fatty acid binding protein 1, liver (Homo sapiens) ] .
  • the method according to any one of (1-1) to (1- 3) which is a method for detecting the presence or absence of a genetic polymorphism, using change (T- ⁇ A) of the 94th amino acid of FABPl as an index.
  • (1-5) The method according to any one of (1-1) to (1- 4), wherein the lipid metabolism disorder or disease caused thereby is a hepatic fatty acid beta-oxidation ability disorder or disease caused thereby.
  • (1-6) The method according to (1-5), wherein the disease caused by the hepatic fatty acid beta-oxidation ability disorder is non-alcoholic steatohepatitis, fatty liver or myocardial infarction.
  • a method for detecting a lipid metabolism disorder or a risk of disease caused thereby comprising the following steps (a) to (c) :
  • a method for detecting, in a subject, a lipid metabolism disorder or a risk of a disease caused thereby comprising the following steps (i) to (iii) : (i) administering a radioisotope-labeled fatty acid to the subject, and detecting, over time, radioactivity generated from the radioisotope-labeled fatty acid accumulated in the liver;
  • step (ii) calculating a fatty acid clearance rate (%/min) in the liver from the change in the radioactivity detected over time in step (i) ;
  • step (iv) The method according to (1-9), further comprising the following step (iv) : (iv) if the fatty acid clearance rate is less than 0.5%/min, determining that the subject has a lipid metabolism disorder or a risk of a disease caused thereby; and if the fatty acid clearance rate is not less than 0.5%/min, determining that the subject has no lipid metabolism disorder or no risk of a disease caused thereby.
  • a labeled or unlabeled 15- to 35-base oligonucleotide which hybridizes with a continuous oligo- or polynucleotide of 16 or more bases in the base sequence of SEQ ID NO: 1 contained in the FABP 1 gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the base sequence is replaced with "u") , the continuous oligo- or polynucleotide containing the 301st nucleotide of the base sequence, and which is used for specifically amplifying the continuous oligo- or polynucleotide.
  • a primer comprising the labeled or unlabeled oligonucleotide according to (1-11), the primer being used for determining a lipid metabolism disorder or a risk of a disease caused thereby.
  • the primer according to (1-12) which is used for identifying the 301st nucleotide of the base sequence of SEQ ID NO: 1 in the FABPl gene on human chromosome 2.
  • a labeled or unlabeled 16- to 500-base oligo- or polynucleotide which hybridizes with a continuous 16- to 500 base oligo- or polynucleotide in the base sequence of SEQ ID NO: 1 contained in the FABP 1 gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the base sequence is replaced with "u") , the continuous oligo- or polynucleotide containing the 301st nucleotide of the base sequence.
  • the probe comprising the labeled or unlabeled oligo- or polynucleotide according to (1-14) , the probe being used for determining a lipid metabolism disorder or a risk of a disease caused thereby. » (1-16).
  • the probe according to (1-15) which is used for identifying the 301st nucleotide of the base sequence of SEQ ID NO: 1 in the FABPl gene on human chromosome 2.
  • a reagent for detecting a lipid metabolism disorder or a risk of a disease caused thereby the reagent comprising the primer according to (1-12) or (1-13) and/or the probe according to any one of (1-15) to (1-17); or a kit comprising at least one of the reagent.
  • NASH non-alcoholic steatohepatitis
  • NASH patients are divided into those with normal hepatic fatty acid beta- oxidation ability (normal hepatic fatty acid beta-oxidation ability-type) and those genetically (inherently) having a low hepatic fatty acid beta-oxidation ability (hepatic fatty acid beta-oxidation ability disorder-type) .
  • NASH non-alcoholic steatohepatitis
  • the present invention provides a method for determining in NASH patients, whether or not the NASH is of hepatic fatty acid beta-oxidation ability disorder-type.
  • a method for detecting NASH of hepatic fatty acid beta-oxidation ability disorder-type comprising the step of detecting, in a biological sample from a subject, a genetic polymorphism that causes change (T->A) of the 94th amino acid of
  • a method for determining whether or not NASH is of hepatic fatty acid beta-oxidation ability disorder-type comprising the following steps (a) to (c) : (a) extracting genomic DNA from a biological sample of a subject;
  • a method for determining whether or not NASH in a subject is of hepatic fatty acid beta-oxidation ability disorder-type comprising the following steps (i) to (iii) :
  • step (ii) calculating a fatty acid clearance rate (%/min) in the liver, from the change in the radioactivity detected over time in step (i) ; and 0 (iii) determining whether the fatty acid clearance rate is less than 0.5%/min or not less than 0.5%/min.
  • a primer comprising the following labeled or 0 unlabeled oligonucleotide, the primer being used for determining whether or not NASH is of hepatic fatty acid beta-oxidation ability disorder-type: a labeled or unlabeled 15- to 35-base oligonucleotide which hybridizes with a continuous oligo- or polynucleotide of 16 5 or more bases in the base sequence of SEQ ID NO: 1 contained in the FABP 1 gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the base sequence is replaced with "u”) , the continuous oligo- or polynucleotide containing the 301st nucleotide of the base sequence, and which 0 is used for specifically amplifying the continuous oligo- or polynucleotide .
  • the primer according to (2-9) which is used for identifying the 301st nucleotide of the base sequence of SEQ ID NO: 1 in the FABPl gene on human chromosome 2.
  • a probe comprising the following labeled or unlabeled oligo- or polynucleotide, the probe being used for determining whether or not NASH is of hepatic fatty acid beta- oxidation ability disorder-type: a labeled or unlabeled 16- to 500-base oligo- or polynucleotide which hybridizes with a continuous 16- to 500-base oligo- or polynucleotide in the base sequence of SEQ ID NO: 1 contained in the FABPl gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the base sequence is replaced with "u”) , the continuous oligo- or polynucleotide containing the 301st nu
  • the probe according to (2-11) which is used for identifying the 301st nucleotide of the base sequence of SEQ ID NO: 1 in the FABPl gene on human chromosome 2. (2-13). The probe according to (2-11) or (2-12), which is immobilized on a solid phase.
  • a reagent for detecting NASH of hepatic fatty acid beta-oxidation ability disorder-type comprising the primer according to (2-9) or (2-10) and/or the probe according to any one of (2-11) to (2-13) ; or a kit comprising the reagent .
  • a method of reducing the risk of developing a lipid metabolism disorder-related disease comprising returning the change (T ⁇ A) in the 94th amino acid of FABPl to the normal state (A ⁇ T) , or returning the change (A-»G) of the base of SNP3513 to the normal state (G->A) , in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G) .
  • An agent for preventing the development of or alleviating NASH in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G) the agent comprising a fibrate drug as an active ingredient.
  • a method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease comprising the following steps of: (a) contacting a test substance with a cell expressing variant FABPl [FABPl (T94A) ] in which the 94th amino acid residue is alanine;
  • step (c) selecting, as a candidate substance, a test substance contacted with the cell expressing variant FABPl to thereby increase the expression level of FABPl or FABPl activity or decrease the expression level of variant FABPl measured in step (b) compared to a negative control, which is the expression level of FABPl or variant FABPl, or FABPl activity of the cell measured in the absence of the test substance.
  • a method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease comprising the following steps of:
  • Method for evaluating therapeutic effects or disease risk reducing effects on lipid metabolism disorders or lipid metabolism disorder-related diseases (5-1) .
  • step (b) determining hepatic fatty acid metabolic ability based on the change in the radiation detected over time in step (a).
  • a method for evaluating a therapeutic effect or a disease risk reducing effect of a treatment on lipid metabolism disorder or lipid metabolism disorder-related disease in a patient with a lipid metabolism disorder caused by having a genetic polymorphism causing change (T-»A) in the 94th amino acid of FABPl as a homozygote comprising the following steps of:
  • step (ii) calculating a fatty acid clearance rate (%/min) in the liver based on the change in the radiation detected over time in step (i) ;
  • Fig. 1 shows the results of 123 I-BMIPP scintigraphy performed in Example 1 (1) . More specifically, Fig. 1 shows images obtained by measuring the amount of gamma rays in (1) the heart (the entire left ventricle), (2) the liver (S8 of the right hepatic lobe) over time (1, 5, 10, 30 minutes) .
  • Fig. 2 shows time-BMIPP retention curves showing hepatic 123 I-BMIPP retention (%) in non-alcoholic steatohepatitis (NASH) patients (37 persons) from immediately after to 1830 seconds after the administration of 123 I-BMIPP (Example 1 (I)).
  • the patients are clearly divided into two groups: a group of patients showing a 123 I-BMIPP clearance rate of less than 0.5 % min (average of 0.27 %/min) ( ⁇ ) , and a group of patients showing a 123 I-BMIPP clearance rate of at least 0.5 %/min. (average of 0.79 %/min.) (O) .
  • Fig. 3 shows the relationship between the 123 I-BMIPP clearance rate (%/min) and the genotype at the 3513rd position of the FABPl gene (polymorphism of the 94 th amino acid of FABPl) in non-alcoholic steatohepatitis (NASH) patients (37 persons) (Example 1 (2)).
  • NASH non-alcoholic steatohepatitis
  • AA represents patients with homozygous G/G as a polymorphism (SNP3513) that causes a 94th amino acid change (T ⁇ A) in FABPl
  • AT represents patients with heterozygous A/G or G/A as a polymorphism (SNP3513) that causes a 94th amino acid change (T-»A) in FABPl
  • TT represents patients with homozygous A/A (patients with TT as the 94 th amino acid of FABPl) .
  • Fig. 4 shows time-BMIPP retention curves otained by measurements before ( ⁇ ) the administration of fibrate drug to FABPl variant NASH patients and after (O) the administration in Example 1 (3) .
  • Fig. 5 shows a diagram showing the relationship between 123 I-BMIPP clearance rate (%/min) and body mass index in nonalcoholic steatohepatitis (NASH) patients (37 persons) (Example 3).
  • Fig. 6 shows the relationship between body mass index
  • BMI liver biopsy- specimens from non-alcoholic steatohepatitis (NASH) patients (37 persons) (Example 3) .
  • Fig. 7 shows the results of measuring liver fat accumulation in tamoxifen-induced NASH patients by CT before (the upper diagram) and after (the lower diagram) 1 year of Bezafibrate administration (Example 3) .
  • Fig. 8 shows time-BMIPP retention curves obtained by measurements before ( ⁇ ) and after (O) 1 year of Bezafibrate administration (Example 3) .
  • nucleotide sequences nucleic acids, amino acids, etc. are those recommended by IUPAC and IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem. , 138, 9 (1984)], and "Guidelines for the preparation of specifications which contain nucleotide and/or amino acid sequence” (Japanese Patent Office) , and those conventionally used in the art.
  • the term “gene” encompasses double-stranded DNA including human genome DNA, and single-stranded DNA (sense strand) and single-stranded DNA that has a sequence complementary to the sense strand (antisense strand) , and fragments thereof. Further, unless otherwise stated, “gene” is used to indicate any of control regions, coding regions, exons and introns. In this specification and the appended claims,
  • Nucleotide has the same meaning as “nucleic acid”, and encompass both DNA and RNA. These may be double- or single-stranded, and a "nucleotide” (or “oligonucleotide” or “polynucleotide”) with a certain sequence encompasses a “nucleotide” (or “oligonucleotide” or
  • polynucleotide with a sequence complementary to the sequence. Further, when the “nucleotide” (or “oligonucleotide” or “polynucleotide”) is RNA, the base “t” in the base sequence is replaced with “u”. In this specification and the appended claims, “Genetic polymorphism” indicates two or more genetically determined alleles.
  • Genetic polymorphism encompasses both single nucleotide polymorphisms (SNPs) , which are caused by a variation of a single nucleotide, and multiple nucleotide polymorphisms extending over two or more continuous nucleotides. Polymorphisms occur at a frequency of at least 1%, and preferably at least 10% in a selected population.
  • lipid metabolism disorder susceptibility gene means a gene that determines susceptibility to a lipid metabolism disorder and diseases caused thereby.
  • the target lipid metabolism disorder of the present invention is preferably a fatty acid beta-oxidation ability disorder in an organ, tissue or cell that expresses hepatic FABPl (fatty acid binding protein 1 * liver) , and more preferably a fatty acid beta- oxidation ability disorder in the liver.
  • a "fatty acid beta- oxidation ability disorder” means a dysfunction of ability to perform fatty acid beta-oxidation. For example, after ingestion of a high-fat diet, fatty acid beta-oxidation ability is compensatorily enhanced in the normal condition, but with a fatty acid beta-oxidation ability disorder, no or extremely little such compensatory enhancement takes place.
  • Nonalcoholic steatohepatitis encompasses tamoxifen-induced non-alcoholic steatohepatitis and other types of non-alcoholic steatohepatitis
  • fatty liver encompasses non-alcoholic fatty liver and non-obese fatty liver.
  • gene frequency refers to the frequency of one allele among all alleles at a gene locus in a population.
  • linkage disequilibrium means a situation in which a combination of alleles located close to each other occurs in a population more frequently than expected to occur by coincidence. For example, when locus X has alleles a and b (which are present at equal frequencies) and locus Y in the vicinity has alleles c and d (which are present at equal frequencies), haplotype ac, i.e., another combination of genetic polymorphisms, is expected to be present at a frequency of 0.25 in the population. If haplotype ac is present at a frequency greater than the expected value, i.e., when the specific genotype ac occurs frequently, alleles a and c are said to be in linkage disequilibrium.
  • Linkage disequilibrium is generated because a specific combination of alleles is naturally selected or introduced into a population recently from an evolutionary standpoint, and can be caused when the linked alleles do not equilibrate.
  • types of linkage disequilibrium differ among different populations, such as nations or races. That is, alleles a and c may be in linkage disequilibrium in one population, while alleles a and d may be in linkage disequilibrium in another population. Detection of genetic polymorphisms in linkage disequilibrium is effective for detecting susceptibility to a disease, although such polymorphisms do not directly cause the disease.
  • allele a at locus X may indicate susceptibility to a disease through linkage disequilibrium with allele c at locus Y, although allele a is not a genetic factor of the disease.
  • linkage disequilibrium analysis means an analysis of the degree of linkage disequilibrium in a genomic region. The sequences and positional information of proteins and genes described in this specification and the appended claims are based on the NCBI (National Center for Biotechnology Information, USA) databases.
  • fatty acid-binding protein 1 [fatty acid-binding protein 1, liver (Homo sapiens) : FAPBl] gene (FAPBl gene) that controls intracellular transportation and metabolism of fatty acids is known to have a plurality of SNPs (e.g., MutDB, http://mutdb.org/AnnoSNP/data/N3/S0/DA/AC.nt.html; and
  • non-alcoholic steatohepatitis can be divided into two types: those in whom hepatic fatty acid beta-oxidation ability is compensatorily enhanced after ingestion of a high-fat diet (normal hepatic fatty acid beta- oxidation-type) , and those in whom such compensatory enhancement does not occur (hepatic fatty acid beta-oxidation disorder-type) ; and that patients of hepatic fatty acid beta-oxidation disorder- type frequently have one of the known SNPs of the FABPl gene as a homozygotic polymorphism.
  • NASH non-alcoholic steatohepatitis
  • the SNP is located in exon 2 (the exon 2 region from 3474 to 3566 bp) of the FABPl gene (Genbank Accession No. NMJD01443, total length: 5069 bp) at locus 2pll on human chromosome 2.
  • the 3513rd base of the FABPl gene is changed from a to g (hereinafter this change is sometimes referred to as "a3513g") , with the result that the 94th amino acid residue of FABPl (protein), i.e., a product of the gene, is changed from threonine (T) to alanine (A) (hereinafter this change is sometimes referred to as "T94A”) .
  • the present inventors confirmed that substantially 100% of subjects with steatohepatitis who have a homozygotic polymorphism (a3513g) (genetic change) of the FABPl gene have insufficient hepatic fatty- acid beta-oxidation ability; and are sure that the FABP 1 gene change (a3513g) and the FABPl change (T94A) are extremely closely associated with lipid metabolism disorders due to insufficient fatty acid beta- oxidation ability.
  • the present inventor conducted research on patients with myocardial infarction, and confirmed that such patients also characteristically have the above homozygotic polymorphism (a3513g) (genetic change) of the FABPl gene.
  • the present inventors believe that the FAPB 1 gene change (a3513g) and FABPl change (T94A) are extremely closely associated with lipid metabolism disorders not only of the liver but also of the entire body, and such changes serve as markers for genetic predisposition to lipid metabolism disorders and diseases caused thereby.
  • the present invention provides use of the human FABPl gene (Genbank Accession No. NM_001443, 5069 bp) as a lipid metabolism disorder susceptibility gene.
  • the base sequence of the FABPl gene (mRNA) is shown in SEQ ID NO: 4 (in which t is replaced with u) .
  • the FABPl gene is located at locus 2pll (ranging from positions 88261772 to 88266840) on human chromosome 2 (Genbank Accession No. NM_000002) .
  • Lipid metabolism disorder susceptibility genes include not only the FABPl gene but also genes that are located close to the FABPl gene and are in strong linkage disequilibrium with the FABP 1 gene, such as, for example, the P gene and R gene.
  • preferable lipid metabolism disorder susceptibility genes are those overlapping "haplotype blocks", in which linkage disequilibrium is observed by linkage disequilibrium analysis of markers for lipid metabolism disorder susceptibility SNPs in human chromosome 2 (preferably locus 2pll) , such haplotype blocks including markers for lipid metabolism disorder susceptibility SNPs.
  • “Genes overlapping haplotype blocks” encompasses both genes having the same base sequence as a part of a haplotype block, and genes having the same base sequence as the entire region of a haplotype block.
  • Candidates for such lipid metabolism disorder susceptibility genes include the following, of which preferable are the LOC51315 gene (hypothetical protein LOC51315) , SMYDl gene (SET and MYND domain containing protein 1), FLJ10916 gene (hypothetical protein FLJ10916) and MRPL45Pone gene
  • SIAT9 sialyltransferase 9 (CMP-NeuAc:lactosylceramide alpha-2,3- sialylransferase 9)
  • POLRlA polymerase (RNA) I polypeptide A, 194kDa FLJ20758 : FLJ20758 protein
  • IMMT inner membrane protein
  • MRPL35 mitochondrial ribosomal protein
  • C2orf23 chromosome 2 open reading frame
  • JMJDlA jumonji domain containing IA
  • VPS24 vacuolar protein sorting 24 (yeast)
  • RNFl03 ring finger protein 103
  • FLJ13910 hypothetical protein FLJ13910
  • CD8A CD8 antigen
  • LOC400965 hypothetical L0C400965
  • CD8B1 CD8 antigen, beta polypeptide 1 (p37) MGC4677 :
  • PAFAHlPl platelet-activating factor acetylhydrolase, isoform Ib, pseudogene 1 PLGL : plasminogen-like
  • LOC51315 hypothetical protein
  • MRPL45P1 mitochondrial ribosomal puotein L45 pseudogene 1
  • FLJ25369 hypothetical protein FLJ25369
  • EIF2AK3 eukarytic translation initiation factor 2-alpha kinase 3
  • RPIA ribose 5-phosphate isomerase A (ribose 5-phosphate epimerase)
  • IGKV4-1 immunoglobulin kappa variable 4-1
  • IGKV7-3 immunoglobulin kappa variable 7-3
  • IGKV2-4 immunoglobulin kappa variable 2-4
  • IGKV3-7 immunoglobulin kappa variable 3-7
  • IGKV2-10 immunoglobulin kappa variable 2-10 IGKV2-14 immunoglobulin kappa variable 2-14 IGKV3-15 immunoglobulin kappa variable 3-15 IGKV2-18 immunoglobulin kappa variable 2-18 IGKV3-20 immunoglobulin kappa variable 3-20 IGKV6-21 immunoglobulin kappa variable 6-21 IGKV2-23 immunoglobulin kappa variable 2-23 IGKV2-24 immunoglobulin kappa variable 2-24 IGKV2-25 immunoglobulin kappa variable 2-25 IGKV2-26 immunoglobulin kappa variable 2-26 IGKV2-28 immunoglobulin kappa variable 2-28 IGKV2-29 immunoglobulin kappa variable 2-29 IGKV2-30 immunoglobulin kappa variable 2-30 IGKV3-31 immunoglobulin kappa variable 3-31 IGKV3-34 immunoglobulin kappa
  • Diagnostic marker for lipid metabolism disorder The present invention provides use of a gene (or oligo- or polynucleotide) containing a marker for genetic predisposition to a lipid metabolism disorder and/or diseases caused thereby, as a diagnostic marker for a lipid metabolism disorder and disease caused thereby.
  • lipid metabolism disorders include hepatic fatty acid beta-oxidation ability disorders.
  • "Fatty acid beta-oxidation ability disorder” means a condition in which, for example, after ingestion of a high-fat diet, compensatory increase in fatty acid toeta-oxidation ability, which takes place in normal conditions, does not occur or occurs only to a slight degree.
  • lipid metabolism disorders lipid metabolism disorder-related diseases
  • hepatic fatty acid beta-oxidation ability disorders examples include hepatic fatty acid beta-oxidation disorders, non-alcoholic steatohepatitis (including tamoxifen-induced nonalcoholic steatohepatitis and other types of non-alcoholic steatohepatitis) , and non-obese fatty liver (including non-obese fatty liver and non-alcoholic fatty liver) .
  • lipid metabolism disorder or lipid metabolism disorder-related disease In individual subjects, the presence or absence of a lipid metabolism disorder or lipid metabolism disorder-related disease, or a risk (potential possibility of development) of such a disease can be detected (tested, diagnosed) by detecting, in the subjects, a gene (or an oligo- or polynucleotide) containing a genetic predisposition marker (diagnostic marker) .
  • diagnostic markers include oligo- and polynucleotides that contain at least one lipid metabolism disorder susceptibility SNP existing in a haplotype block, and that are specifically recognized in the human genome.
  • haplotype block is a region in which linkage disequilibrium is observed by linkage disequilibrium analysis of lipid metabolism disorder susceptibility SNPs in human chromosome 2 (preferably locus 2pll) , the region containing a lipid metabolism disorder susceptibility SNP.
  • the length (base length) of such oligo- and polynucleotides is not limited, as long as it is a length specifically recognized in the human genome.
  • the length is usually at least 10 bases, and preferably at least 20 bases.
  • diagnostic markers include oligo- and polynucleotides containing the genetic polymorphism (the 3513rd base (A or T) in the 1st to 5069th bases of the FABPl gene, hereinafter sometimes referred to as "SNP3513") existing in a haplotype block in the base sequence of the FABPl gene (Genbank Sequence Accession IDs:NM_001443, full length: 5069 bp) on human chromosome 2.
  • oligo- and polynucleotides consisting of 51 to 601 bases in the base sequence of the FABPl gene on human chromosome 2 and having, in its middle, the genetic polymorphic site "SNP3513" (the 3513rd base of the FABPl gene) (lipid metabolism disorder susceptibility SNP) [e.g., 51 bases (25 bases from each of the 5' and 3' end sides of SNP3513: SEQ ID NO: 5), 201 bases (100 bases from each of the 5' and 3' end sides of SNP3513: SEQ ID NO: 6), and 601 bases (300 bases from each of the 5' and 3' end sides of SNP3513: SEQ ID NO: 7) ] .
  • SNP3513 the 3513rd base of the FABPl gene
  • SNP3513 lipid metabolism disorder susceptibility SNP
  • gene products e.g., mRNA, its derivative (cDNA) , proteins
  • cDNA its derivative
  • Such diagnostic markers include FABPl (protein) expressed from the FABPl gene, peptides having partial amino acid sequence of FABPl, and oligo- and polynucleotides (mRNA and cDNA) coding for such protein and peptides.
  • SEQ ID NO: 8 shows the unchanged amino acid sequence (127 amino acids) of FABPl (i.e., the 94th amino acid is Thr) and the cDNA sequence (381 bp) coding therefor.
  • the above-mentioned pepetides are oligo- or polypeptides with amino acid sequences containing at least the 94th amino acid of FABPl, which corresponds to the above-mentioned "SNP3513" of the FABPl gene.
  • the present invention also provides a method for detecting in a subject the presence or absence of a lipid metabolism disorder, or the presence or absence of potential possibility of development risk of a disease caused by a lipid metabolism disorder (lipid metabolism disorder-related disease) .
  • the method can be performed by detecting a genetic polymorphism that causes change of the 94th amino acid of FABPl from threonine (T) to alanine (A) (T94A) .
  • T threonine
  • A alanine
  • a polymorphism is generally defined as a variation of base(s) in a gene, occurring at a frequency of at least 1% of a population.
  • polymorphism as used herein is not limited to that definition, and encompasses genetic changes occurring at frequencies less than 1% of a population. Polymorphisms include single nucleotide polymorphisms (SNPs) , multiple nucleotide polymorphisms, which are deficiencies, substitutions or insertions of several tens of bases; etc., and the target polymorphism of the present invention is not limited to a specific type of polymorphism, as long as it causes a change of the 94th amino acid (T ⁇ A) of FAPBl. The number of polymorphisms is also not limited, and two or more polymorphisms may be presented.
  • SNPs single nucleotide polymorphisms
  • T ⁇ A 94th amino acid
  • such genetic polymorphism (s) can be detected by known methods, such as (1) carrying out PCR in a region containing the genetic polymorphism(s) , followed by detection by the SSCP method; (2) carrying out PCR in a region containing the genetic polymorphism (s) , followed by detection by observing the restriction enzyme cleavage pattern of the PCR product; (3) carrying out PCR in a region containing the genetic polymorphism (s) , followed by direct sequencing of the PCR product; (4) the ASO (allele specific oligonucleotide) method in which an oligonucleotide probe that hybridizes with a region containing the genetic polymorphism (s) is hybridized with DNA of an individual; (5) a method in which an oligonucleotide probe that hybridizes with a region containing the genetic polymorphism (s) is used, followed by detection by mass spectroscopy or the like; etc.
  • ASO allele specific oligonucleotide
  • a detection method can be used in which an SNP (a->g) of 3513rd base of the FABPl gene, which is a genetic polymorphism that causes a change (T->A) of the 94th amino acid of FABPl, is detected.
  • This detection method can be performed by the following steps (a) and (b) :
  • step (b) the FABPl gene in the extracted genomic DNA need not be specified, and the base between the base sequences shown in SEQ ID NOS: 2 and 3 is identified in the extracted genomic DNA itself.
  • the base sequence shown in SEQ ID NO: 2 corresponds to the base sequence of 300 bases located at the 5' end side (upstream) of the above-mentioned lipid metabolism disorder susceptibility SNP base (SNP3513) in the FAPBl gene on human chromosome 2 (the 3513th base of the sequence deposited under Genbank Sequence Accession No: NM_001443) .
  • the base sequence shown in SEQ ID NO: 3 corresponds to the base sequence of the 300 bases located at the 3' end side (downstream) of the lipid metabolism disorder susceptibility SNP base (SNP3513) in the base sequence of the FABPl gene on human chromosome 2.
  • the detection target is SNP3513, i.e., a lipid metabolism disorder susceptibility SNP, located between the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3.
  • the subject who provided the genomic DNA sample is deemed to have a lipid metabolism disorder and is likely to develop a disease caused by a lipid metabolism disorder (has a relatively high risk of developing a disease caused by a lipid metabolism disorder) .
  • the SNP 3513 base is homozygous for A (adenine)
  • A/A or heterozygous for A (adenine) and G (guanine) (A/G or G/A) , the subject who provided the genomic DNA sample is deemed not to have a lipid metabolism disorder and is unlikely to develop a disease caused by a lipid metabolism disorder (has a relatively low risk of developing a disease caused by a lipid metabolism disorder) .
  • the method of the present invention further comprises the following steps (c) and (d) :
  • the method of the present invention can determine the presence or absence of a lipid metabolism disorder, and susceptibility or insusceptibility to a lipid metabolism disorder-related disease, i.e., a risk of developing a lipid metabolism disorder-related disease.
  • the risk of developing a lipid metabolism disorder- related disease can be ascertained in a mechanical fashion by- using an A-»G change (homozygotic polymorphism) of SNP3513 as an index for judgment, without need for diagnosis by an expert such as a doctor.
  • the method of the present invention can also be said to be a method for detecting a lipid metabolism disorder or a risk of developing a lipid metabolism disorder-related disease.
  • the sample for genomic DNA extraction in step (a) may be obtained from materials isolated from a subject or clinical specimen, etc., including all kinds of cells (including cultured cells but excluding reproductive cells), tissues (e.g., tissues of the liver, kidney, adrenal gland, uterus, brain and the like, including cultured tissues), body fluids (e.g., saliva, lymph, respiratory tract mucus, semen, sweat, urine and the like) , etc.
  • Preferable materials include leukocytes and mononuclear cells isolated from peripheral blood, of which leukocytes are most preferable. Such materials can be isolated by methods conventionally used in clinical trials.
  • leucocytes when using leucocytes as a material, leucocytes are separated in a standard manner from peripheral blood isolated from a subject. Subsequently, Proteinase K and sodium dodecyl sulfate (SDS) are added to the obtained leukocytes to degrade and denature proteins, and then phenol/chloroform extraction is performed to obtain genomic DNA (or RNA) . RNA can be removed by RNase if necessary.
  • the method for genomic DNA extraction is not limited to the above, and may be a method well- known in the art (e.g., Sambrook J. et.
  • DNA containing the FABPl gene on human chromosome 2 or exon 2 thereof may be isolated.
  • DNA can be isolated by, for example, PCR using a primer that hybridizes with the FABPl gene or exon 2 thereof, together with genomic DNA as a template.
  • step (b) from the thus obtained extract containing human genomic DNA, the lipid metabolism disorder susceptibility SNP that is extremely closely associated with a lipid metabolism disorder, i.e., the SNP3513 base described in detail in 1 and 2 above, is detected.
  • the base can also be detected by directly sequencing the FABPl gene on human chromosome 2 isolated from a sample containing human genome DNA, or more preferably exon 2 of the gene, and analyzing the base (A or G) of the 280th base of exon 2 of the gene (corresponding to the 3513th base of the base sequence deposited under Genbank Sequence Accession No: NM_001443) .
  • the following methods can be employed to identify the target base: in the case where the polymorphic sequence is a restriction site, a method in which the genotype is determined using differences in restriction enzyme cleavage patterns (hereinafter referred to as RFLP) ; a method based on hybridization using a polymorphism-specific probe, for example, a method in which specific probes are attached to glass slides or nylon films, and differences in hybridization strength to these probes are detected to determine the type of polymorphism or the amounts of these probes degraded by polymerase during amplification of the temperate double-strands are detected to evaluate the hybridization efficiency of the specific probes, thereby determining the genotype; a method in which temperature-dependent changes of fluorescence emitted from certain types of double-strand-specific fluorochrome are followed to detect differences in melting temperatures of the double strands, thereby specifying the polymorphism; a method in which complementary sequences are appended to the ends of
  • the above are typical genetic polymorphism detection methods, and the method for detecting a lipid metabolism disorder and/or a risk of developing a lipid metabolism disorder-related disease according to the present invention can utilize not only the above methods but also a wide variety of other genetic polymorphism detection methods presently known or to be developed in the future. In the genetic polymorphism detection according to the present invention, such genetic polymorphism detection methods can be used singly or in combination.
  • a simple method for identifying SNP3513 is a RFLP (restriction enzyme cleavage fragment length polymorphism) method using a HindIII restriction enzyme, in which an oligonucleotide with the base sequence shown in SEQ ID NO: 11 is used as a forward primer and an oligonucleotide with the base sequence shown in SEQ ID NO: 12 as a reverse primer (see the Examples) .
  • RFLP restriction enzyme cleavage fragment length polymorphism
  • the method of the present invention is not limited to direct detection of the presence or absence of the genetic polymorphism, and encompasses detection of the 94th amino acid change (T->A) resulting from the genetic polymorphism.
  • methods for such detection include an analysis of whether or not the above amino acid has been changed in an expression product (e.g., mRNA, FABPl protein) of the FABPl gene of the subject.
  • Such an analysis can be carried out, not only by determining the sequence of an expression product of the FABPl gene, but also by using an antibody to FABPl or variant FABPl (T94A) to perform Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence technique, or by detecting activity caused (or eliminated) by the 94th amino acid change (T ⁇ A) in FABPl.
  • T94A an antibody to FABPl or variant FABPl
  • the above-mentioned method is useful for discerning whether a patient with non-alcoholic steatohepatitis (NASH) or a subject with a potential risk of NASH has insufficient (lowered) hepatic fatty acid beta-oxidation ability, or has normal hepatic fatty acid beta-oxidation ability.
  • NASH non-alcoholic steatohepatitis
  • the present invention therefore provides a method of detecting and identifying in a patient with NASH whether the patient is of lipid metabolism disorder-type (hepatic fatty acid beta-oxidation disorder-type) or of normal lipid metabolism-type (normal hepatic fatty acid beta-oxidation-type) .
  • the method of detecting whether or not a patient with lipid metabolism disorder-type hepatic fatty acid beta-oxidation disorder-type
  • normal lipid metabolism-type normal hepatic fatty acid beta-oxidation-type
  • NASH or a subject with a potential risk of NASH is of lipid metabolism disorder-type (hepatic fatty acid beta-oxidation disorder-type) can be performed in the same manner as above, by detecting the presence or absence of a genetic polymorphism that causes the 94th amino acid change (T-»A) in FABPl, using a biological sample from the patient or subject.
  • lipid metabolism disorder-type hepatic fatty acid beta-oxidation disorder-type
  • the patient or subject is deemed to be of lipid metabolism disorder-type (hepatic fatty acid beta-oxidation ability disorder-type) , for example when the 3513rd base (SNP3513) of the FABPl gene contained in genomic DNA is homozygous for G (G/G) , and to be of normal lipid metabolism-type (normal hepatic fatty acid beta-oxidation ability-type) , for example when the base is homozygous for A (A/A) or heterozygous for A and G (A/G or G/A) .
  • lipid metabolism disorder-type hepatic fatty acid beta-oxidation ability disorder-type
  • SNP3513 3513rd base
  • normal lipid metabolism-type normal hepatic fatty acid beta-oxidation ability-type
  • the fatty acid beta-oxidation ability in the liver can be artificially or forcibly compensated (for example by administrating a medicine that enhances hepatic fatty acid beta- oxidation ability) to ameliorate the symptoms of NASH.
  • the hepatic fatty acid beta- oxidation ability can be artificially or forcibly compensated (for example by administering a medicine that enhances hepatic fatty acid beta-oxidation ability) to inhibit or prevent development of NASH.
  • Medicines that enhance fatty acid beta- oxidation ability include medicines stimulating a PPRE (peroxisome proliferetor response element) located upstream of the FABPl gene, via an in-vivo substance bound to the PPRE, thereby enhancing the production of FABPl (or variant FABPl (T94A) ) , such as, for example, fibrate drugs (e.g., clofibrate, aluminium clofibrate, simfibrate, clinofibrate, bezafibrate) .
  • PPRE peroxisome proliferetor response element
  • fibrate drugs can be effectively used for subjects having in the FABPl gene SNP3513 that is homozygous for G (G/G) , as a medicine for alleviating NASH or preventing the development of NASH of lipid metabolism disorder-type (hepatic fatty acid beta-oxidation disorder-type) .
  • reagents for detecting a lipid metabolism disorder or a susceptibility to (a risk of developing of) a lipid metabolism disorder-related disease
  • kit comprising such reagents
  • reagents for determining whether or not a patient with NASH or a subject with a potential possibility of NASH is of lipid metabolism disorder-type (hepatic fatty acid beta-oxidation disorder-type), and a kit comprising such reagents.
  • Reagent for detecting lipid metabolism disorder or risk of lipid metabolism disorder-related disease and a kit comprising the reagent
  • an oligo- or polynucleotide is used which specifically hybridizes with an oligo- or polynucleotide containing the lipid metabolism disorder susceptibility SNP (genetic polymorphism) in the lipid metabolism disorder susceptibility gene existing on human chromosome 2, to thereby detect the SNP.
  • the oligo or polynucleotide to be used is designed as a 16- to 500-base, preferably 20- to 200-base, and more preferably 20- to 50-base oligo- or polynucleotide which hybridizes with a continuous gene region consisting of the above number of bases and containing the SNP in the lipid metabolism disorder susceptibility gene.
  • oligo- or polynucleotide does not significantly cross- hybridize with other DNA under normal hybridization conditions, and preferably stringent hybridization conditions (e.g., conditions described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).
  • the oligo- or polynucleotide preferably has a base sequence complementary to that of the gene region containing the SNP to be detected, but need not be completely complementary as long as it is able to undergo such specific hybridization.
  • oligo- and polynucleotides include those that specifically hybridize with oligo- or polynucleotides containing the lipid metabolism disorder susceptibility SNP (SNP3513) in the lipid metabolism disorder susceptibility gene (FABPl gene) on human chromosome 2, to thereby detect the SNP.
  • SNP3513 lipid metabolism disorder susceptibility SNP
  • FABPl gene lipid metabolism disorder susceptibility gene
  • a 16- to 500-base oligo- or polynucleotide can be used which hybridizes with a continuous 16- to 500-base oligo- or polynucleotide in the base sequence of SEQ ID NO: 1 contained in the FABP 1 gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the nucleotide sequence is replaced with "u") , the continuous oligo- or polynucleotide containing the 301st nucleotide of the base sequence.
  • the oligo- or polynucleotide is designed as an oligo- or polynucleotide "probe" which specifically hybridizes with an oligonucleotide containing the lipid metabolism disorder susceptibility SNP (SNP3513) in the FABPl gene on human chromosome 2, in order to detect in a subject the presence or absence of a lipid metabolism disorder or a risk of a disease caused thereby (a lipid metabolism disorder-related disease) .
  • Such oligo- and polynucleotides can be synthesized in a standard manner based on the base sequence of the FABPl gene, using, for example, a commercially available nucleotide synthesizer.
  • the probe is labeled with a radioactive substance, fluorescent substance, chemiluminescent substance, enzyme or the like so that an oligonucleotide containing SNP3513 can be detected.
  • the above probe may also be used immobilized on a solid phase.
  • the present invention also provides the above probe (oligo- or polynucleotide) as an immobilized probe (e.g., a gene chip, cDNA microarray, oligo DNA array, membrane filter or the like on which the probe is immobilized) .
  • the probe can be advantageously used for a DNA chip for detecting a lipid metabolism disorder susceptibility gene.
  • the solid phase to be used for such immobilization is not limited as long as it can immobilize the oligo- or polynucleotide, and may be, for example, a glass plate, nylon membrane, micro bead, silicon chip, capillary or other substrate.
  • the oligo- or polynucleotide may be first synthesized and then immobilized on a solid phase, or may be synthesized on a solid phase.
  • a commercially available spotter e.g., a product of Amersham
  • This and other immobilization methods are well-known in the art and can be selected according to the type of immobilized probe to be obtained [e.g., in situ oligonucleotide synthesis by photolithographic techniques
  • an oligonucleotide of about 20 bases that is complementary to a region containing the lipid metabolism disorder susceptibility SNP (SNP3513) is designed as a probe.
  • That probe is labeled with a fluorochrome at the 5' end and with a quencher at the 3' end, and specifically hybridizes with sample DNA, but does not itself emit luminescence, and is detected by the fluorochrome released by cleavage of the fluorochrome binding to the 5' end by an extension reaction using a separately added upstream PCR primer.
  • oligonucleotides complementary to two sequences (3' and 5' end sides) adjacent to the polymorphic site are designed as probes. Detection is carried out by using a third probe that is unrelated to these two probes.
  • the present invention also provides an oligonucleotide for use as a primer for specifically amplifying a sequence region containing a lipid metabolism disorder susceptibility SNP in a lipid metabolism disorder susceptibility gene (a genetic polymorphism, for example, SNP3513 in the FABPl gene) on human chromosome 2.
  • a lipid metabolism disorder susceptibility gene a genetic polymorphism, for example, SNP3513 in the FABPl gene
  • Such a primer is designed as an oligonucleotide of about 15 to about 35 bases, and preferably about 18 to about 30 bases, that specifically hybridizes with a part of a continuous oligo- or polynucleotide containing the nucleotide at the lipid metabolism disorder susceptibility SNP site in the lipid metabolism disorder susceptibility gene, and thereby specifically amplifies the continuous oligo- or polynucleotide.
  • the length of the oligo- or polynucleotide to be amplified is suitably selected according to the detection method to be employed, and is usually 15 to 1000 bases, preferably 20 to 500 bases, and more preferably 20 to 200 bases.
  • primers include an oligonucleotide of about 15 to about 35 bases, and preferably about 18 to about 30 bases, that specifically hybridizes with a part of a continuous oligo- or polynucleotide containing the nucleotide at the SNP
  • oligonucleotides include those that specifically hybridize with an oligo-r or polynucleotide containing the lipid metabolism disorder susceptibility SNP (SNP3513) in the lipid metabolism disorder susceptibility gene (FABPl gene) on human chromosome 2 to thereby detect the SNP.
  • SNP3513 lipid metabolism disorder susceptibility SNP
  • FABPl gene lipid metabolism disorder susceptibility gene
  • oligonucleotide of about 15 to about 35 bases, and preferably about 18 to 30 about bases, that hybridizes with and thereby specifically amplifies a continuous oligo- or polynucleotide of 16 bases or more in the base sequence of SEQ ID NO: 1 contained in the FABP 1 gene on human chromosome 2 (when the oligo- or polynucleotide is RNA, the base "t" in the base sequence is replaced with "u”) , the continuous oligo- or polynucleotide containing the 301st nucleotide of the base sequence .
  • Such oligonucleotides can be synthesized in a standard manner based on a known base sequence of a lipid metabolism disorder susceptibility gene, such as the FABPl gene, using a commercially available nucleotide synthesizer.
  • MALDI-TOF/MS Microx-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry
  • the primer is hybridized with sample DNA immobilized on a silicon chip, and ddNTP is added to extend the DNA sequence by a single base. Subsequently, the extended DNA sequence is separated, and a polymorphism is detected by mass spectrometry.
  • the primer usually consists of at least 15 bases, and is preferably designed to be as short as possible.
  • the probe and primer of the present invention may be labeled with a substance suitable for detection of a genetic polymorphism, such as, for example, a fluorochrome, enzyme, protein, radioisotope, chemiluminescent substance, biotin or the like.
  • a substance suitable for detection of a genetic polymorphism such as, for example, a fluorochrome, enzyme, protein, radioisotope, chemiluminescent substance, biotin or the like.
  • Fluorochromes preferably usable in the present invention include those generally used for labeling nucleotides for detection or quantitation of nucleic acids.
  • flurochromes examples include, but are not limited to, HEX (4, 7, 2' , 4' , 5' , 7'-hexachloro-6-carboxyfluorescein, a green fluorochrome) , fluorescein, NED (tradename of Applied Biosystems, a yellow fluorochrome) , 6-FAM (tradename of Applied Biosystems, a yellow- green fluorochrome), rhodamine and derivatives thereof [e.g., tetramethyl rhodamine (TMR) ] , etc.
  • Methods for labeling nucleotides with fluorochromes can be suitably selected from known methods [Nature Biotechnology, 14, 303-308 (1996)].
  • fluorescent-labeling kits are also usable (e.g., the oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia) .
  • the primer of the present invention may contain a linker sequence attached to one end thereof for polymorphism detection.
  • linker sequences include flaps (sequences that are completely unrelated to sequences in the vicinity of the polymorphism) added to the 5' end of the oligonucleotide, as used in the invader method described above.
  • preferable examples of forward primers for detecting a base sequence containing the lipid metabolism disorder susceptibility SNP (SNP3513) in the FABPl gene on human chromosome 2 include, as well as oligonucleotides having the base sequence shown in SEQ ID NO: 11, oligonucleotides having a continuous sequence of 15 bases or more (preferably 15 to 35 bases) that hybridize with the region from the 1st to 300th base of the base sequence shown in SEQ ID NO: 7; and preferable examples of reverse primers include, as well as oligonucleotides having the base sequence shown in SEQ ID NO: 12, oligonucleotides having a continuous sequence of 15 bases or more (preferably 15 to 35 bases) that hybridize with the region from the 302nd to 601st base of the base sequence shown in SEQ ID NO: 7.
  • the above probe and primer, which may be labeled, can be
  • the present invention further provides a kit comprising a reagent for detecting a lipid metabolism disorder or or a risk of developing a lipid metabolism disorder-related disease.
  • the kit comprises at least one oligo- or polynucleotide (which may be labeled and/or immobilized on a solid phase) that can be used as the above-mentioned probe or primer.
  • the kit of the present invention may comprise, in addition to the probe or primer, other reagents, devices and the like that are required for carrying out the method of the present invention, such ,as hybridization reagents, probe labels, agents for detecting labels, buffer solutions, etc.
  • the invention shows that the 94th amino acid change (T->A) in the amino acid sequence of the fatty acid binding protein 1 (FABPl) closely relates to human lipid metabolism disorder. Based on this finding, by reversing the amino acid change to its natural (normal) state (A—»T) or by restoring the reduction of FABPl function caused by the amino acid change (for example, disorder or reduction of the compensatory hyperfunction of fatty acid ⁇ -oxidation ability) to its natural state, lipid metabolism disorder can be alleviated and the risk of developing lipid metabolism disorder-related disease can be reduced.
  • the amino acid change (T94A) of FABPl closely relates to the change (A—>G) of the nucleotide at position 3513 of the lipid metabolism disorder susceptibility SNP (SNP3513) (nucleotide at position 3513 of the sequence of Genbank Sequence Accession IDs: NM_001443) located in the FABPl gene of human chromosome 2. Based on this, also by restoring the nucleotide change (A ⁇ G) at position 3513 of SNP to its natural state (G ⁇ A) , lipid metabolism disorder can be alleviated and the risk of developing lipid metabolism disorder-related disease can be reduced.
  • SNP3513 lipid metabolism disorder susceptibility SNP
  • the present invention also provides a method for reducing the risk of developing lipid metabolism disorder-related diseases and a component effective for reducing the risk thereof.
  • the method for reducing the risk of developing lipid metabolism disorder-related diseases can be restate as a method for preventing the development of lipid metabolism disorder-related diseases (lipid metabolism disorder- related disease prevention method) and a method for alleviating lipid metabolism disorder-related diseases (lipid metabolism disorder-related disease alleviating method) (The "method for reducing the risk of lipid metabolism disorder-related diseases" of the invention encompasses both the "lipid metabolism disorder- related disease prevention method" and the "lipid metabolism disorder-related disease alleviating method”) .
  • the invention provides a lipid metabolism disorder alleviating agent comprising FABPl or FABPl gene as an active ingredient.
  • the lipid metabolism disorder alleviating agent comprising FABPl as an active ingredient is preferably prepared in a dosage form such that FABPl is transported to the liver and then functions therein.
  • the lipid metabolism disorder alleviating agent comprising FABPl gene as an active ingredient is preferably prepared in a dosage form such that FABPl gene is transported to the liver, then expressed to exhibit its function in the liver.
  • artificial or forcible compensation for hepatic fatty acid ⁇ - oxidation ability also can treat lipid metabolism disorder and also can reduce the risk of developing lipid metabolism disorder-related diseases.
  • agents for increasing fatty acid ⁇ -oxidation ability include fibrate drugs (e.g., clofibrate, aluminium clofibrate, simfibrate, clinofibrate, bezafibrate) as described above.
  • the invention also provides effective methods for developing drugs and foods useful for achieving the above- described method for reducing the risk of developing lipid metabolism disorder-related diseases (method for preventing lipid metabolism disorder-related diseases and method for alleviating lipid metabolism disorder-related diseases) . More specifically, the invention provides a method for selecting a candidate substance (screening method) effective for alleviating lipid metabolism disorders and reducing the risk of developing lipid metabolism disorder-related diseases (for preventing lipid metabolism disorder-related diseases and/or for alleviating lipid metabolism disorder-related diseases) .
  • the screening can be carried out by the following steps of:
  • step (b) measuring the expression level of FABPl or variant FABPl, or FABPl activity with respect to the cell contacted with the test substance; and > (c) selecting, as a candidate substance, a test substance contacted with the cell expressing variant FABPl to thereby increase the expression level of FABPl or FABPl activity, or decrease the expression level of variant FABPl measured in step (b) compared to a negative control, which is the expression level of FABPl or variant FABPl, or FABPl activity of the cell measured in the absence of the test substance.
  • test cell employed in the method may be any cell insofar as the cell expresses and produces variant FABPl [FABPl (T94A) ] in which the 94th amino acid residue is alanine (replacing threonine) ; and the origin of the variant FABPl gene
  • variant FABPl gene (a3513g) may be endogenous or exogenous.
  • the cell expressing exogenous variant FABPl can be produced by introducing into a host cell an expression vector into which a variant FABPl gene (a3513g) usually encoding the protein is incorporated in an expressive manner.
  • the origin of the cell is not restricted within the above limitation [e.g., bacteria (e.g., eukaryotes, such as yeasts) , insect cells, and animal cells, etc.].
  • bacteria e.g., eukaryotes, such as yeasts
  • insect cells and animal cells, etc.
  • preferable are cells derived from an animal, such as human, monkey, mouse, rat, cow, pig, and dog. Such cells can be effectively used as a screening tool in the method for screening for a candidate substance effective for alleviating lipid metabolism disorders and reducing the risk of developing lipid metabolism disorder-related diseases.
  • test substance used in the screening method there is no limitation to the test substance used in the screening method, and for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, and peptides, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microbial products, marine biological extracts, and plant extracts can be mentioned.
  • Contact of the test substance with the cell expressing variant FABPl is, for example, performed by adding the test substance to a culture medium solution of cells expressing variant FABPl, but is not limited to* this method.
  • the test substance is a protein or the like
  • "contact” can be performed by transfecting the cell with a DNA vector constructed to express the protein.
  • the expression level of FABPl and/or variant FABPl, or FABPl activity with respect to the variant FABPl expression cell contacted with the test substance is measured.
  • the gene expression level of FABPl or variant FABPl can be measured by, for example, Western blotting, Dot blotting, Immunoprecipitation assay, Enzyme-linked immunosorbent assay (ELISA), Immunofluorescence method, etc., using antibodies against FABPl or variant FABPl.
  • the measurement methods are not limited to the above.
  • measurement of FABPl activity can be carried out by Scatchard plot analysis using a substance that binds to FABPl, or a binding inhibition experiment or binding enhancement experiment using a substance that binds to FABPl.
  • hydrophobic materials such as fatty acids (e.g., oleic acid), fibrate drugs (e.g., clofibrate, bezafibrate, etc.), bile acid, are known as substances that bind to FABPl.
  • the screening method of the invention can be carried out by the following steps of:
  • Step (2) is not limited to a method for directly evaluating change (g—>a) of the base at position 3513 of the lipid metabolism disorder disease susceptibility SNP (SNP3513) (nucleotide at position 3513 of the sequence of Sequence Accession IDs: NM_001443 (full length 5069bp) ) located in the FABPl gene of human chromosome 2.
  • the base change may be indirectly evaluated by, for example, a method for evaluating a specific protein about its function, activity, or amount, which is produced (increased) or lost (decreased) due to the base change (g-»a) at position 3513 of SNP.
  • Any cell can be employed in Step 1 insofar as FABPl gene on human chromosome 2 or a gene comprising at least exon 2 region of FABPl gene can be introduced thereto, and the gene can be stably held therein.
  • the origin of the cell e.g., bacteria (e.g., eukaryotes, such as yeasts), insect cells, animal cells, etc.) limited. The previously-mentioned animal cells are preferable. Gene transfer, cell culture, etc.
  • Such cells can be effectively used as a screening tool in the method (screening method) for screening for a candidate substance effective for reducing the risk of developing lipid metabolism disorder disease.
  • test substance used in the screening method there is no limitation to the test substance used in the screening method, and for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, and peptides, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microbial products, marine biological extracts, and plant extracts can be mentioned.
  • the screening method of the invention can be carried out by the following steps of:
  • step (i) measuring the binding affinity between variant FABPl and a FABPl-binding substance in the presence of a test substance, and (ii) selecting, as a candidate substance, a test substance that increases the binding affinity between the variant FABPl and the FABPl-binding substance measured in step (i) compared to a negative control, which is binding affinity between the variant FABPl and the FABPl-binding substance measured in the absence of the test substance.
  • FABPl-binding substances used herein.
  • examples of such substances include various hydrophobic materials, such as fatty acids (e.g., oleic acid), fibrate drugs (e.g., bezafibrate, etc.), bile acid, etc.
  • Variant FABPl can be prepared, for example, as follows. mRNA is purified using a commercially-available mRNA purification kit from human liver tissues containing a variant FABPl gene, and subsequently a single stranded DNA corresponding to the mRNA is created using reverse transcriptase. With the single stranded DNA as a template, cDNA containing full length FABPl is created using upstream primer (GGCAGAGCCGCAGGTCAGTCGTG) (SEQ ID NO: 9) and downstream primer (ATAATATGAAATGCAGACTTGTT) (SEQ ID NO: 10), the cDNA thus created is then incorporated in a commercially available protein expression vector, and genetic change human FABPl is created using a commercially available cell-free protein expression kit.
  • upstream primer GGCAGAGCCGCAGGTCAGTCGTG
  • downstream primer ATAATATGAAATGCAGACTTGTT
  • the genetic change human FABPl is purified and obtained using a purification column.
  • the method for measuring binding affinity between variant FABPl and a FABPl-binding substance can be measured based on the binding reaction of a labeled oleic acid, or the like, which is known to bind to FABPl, etc.
  • the binding affinity can be measured by, for example, Western blotting, Dot blotting, Immunoprecipitation assay, Enzyme-linked immunosorbent assay (ELISA), Immunofluorescence method, etc., using antibodies against variant FABPl.
  • the binding affinity of the test substance to the variant FABPl can be measured by contacting a test substance with variant FABPl, and then conducting Scatchard plot analysis using a FABPl-binding substance, or a binding inhibition experiment or binding enhancement experiment using a FABPl-binding substance.
  • a test substance is not necessarily contacted with variant FABPl protein before measurement, and may be contacted therewith at the same time of or after measurement as in a competition experiment.
  • test substance to be used in the screening method there is no particular limitation to the test substance to be used in the screening method, and for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, and peptides, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microbial products, marine biological extracts, plant extracts, etc. can be mentioned.
  • the candidate substance selected in step (c) , (2), or (ii) (candidate substance selecting step) as mentioned above undergoes, if required, a further pharmacological test, clinical trial, or toxicity test, thereby providing an active ingredient more effective and safer for humans for use in an agent for reducing the risk of developing lipid metabolism disorder diseases (agents for preventing lipid metabolism disorder-related diseases, agents for alleviating lipid metabolism disorder- related diseases, health food) .
  • the candidate substance thus obtained is prescribed or formulated into pharmaceutical preparations according to well- known procedures, thereby providing an agent for reducing the risk of developing lipid metabolism disorder-related diseases (agents for preventing lipid metabolism disorder-related diseases, agents for alleviating lipid metabolism disorder-related diseases, or health food) .
  • the invention provides a method for evaluating therapeutic effects and/or disease risk-reducing effects of an agent or other treatment on lipid metabolism disorders caused by genetic polymorphism causing change (T-»A) in the 94th amino acid of FABPl as a homozygote, or diseases caused thereby (lipid metabolism disorder-related diseases) .
  • the method of the invention is characterized by comprising a step of evaluating hepatic fatty acid metabolic ability (hepatic fatty acid ⁇ - oxidation ability) .
  • the invention is based on the following findings.
  • a person who has a polymorphism (a3513g) causing the change (T-»A) in the 94th amino acid of FABPl as a homozygote (G/G) develops with high frequencies fatty acid metabolic disorder, and more specifically, hepatic fatty acid ⁇ -oxidation disorder. Consequently, the person develops lipid metabolism disorder and is likely to suffer from lipid metabolism disorder- related diseases [e.g., lipid-metabolism disorder, non-alcoholic steatohepatitis (tamoxifen-induced non-alcoholic steatohepatitis and the other non-alcoholic steatohepatitis, etc. ) , fatty liver (non-obesity fatty liver, non-alcoholic steatohepatitis) , myocardial infarction, etc.].
  • lipid metabolism disorder-related diseases e.g., lipid-metabolism disorder, non-alcoholic steatohepatitis (
  • hepatic fatty acid ⁇ -oxidation ability of those who have the gene polymorphism causing the change (T->A) in the 94th amino acid of FABPl as a homozygote the lipid metabolism ability inherent to the living body can be compensated to alleviate lipid metabolism disorder (reduction) .
  • lipid metabolism disorders e.g., lipid metabolism disorder, non-alcoholic steatohepatitis (tamoxifen-induced nonalcoholic steatohepatitis, and other non-alcoholic steatohepatitis, etc.), fatty liver (non-obesity fatty liver, non-alcoholic steatohepatitis), myocardial infarction, etc.
  • lipid metabolism disorders e.g., lipid metabolism disorder, non-alcoholic steatohepatitis (tamoxifen-induced nonalcoholic steatohepatitis, and other non-alcoholic steatohepatitis, etc.
  • fatty liver non-obesity fatty liver, non-alcoholic steatohepatitis
  • myocardial infarction etc.
  • such therapeutic effects can be evaluated by comparing hepatic fatty acid metabolic abilities before and after treatment, more specifically fatty acid ⁇ - oxidation abilities, and determining the presence or absence of the enhancement of the abilities after treatment.
  • the method for evaluating therapeutic effects or disease risk reducing effects of the invention can be carried out by the following steps of: (1) administering a radioisotope-labeled fatty acid to a subject with lipid metabolism disorder caused by having a genetic polymorphism causing change (T->A) in the 94th amino acid of FABPl as a homozygote or risk of developing lipid metabolism disorder-related diseases; (2) detecting, over time, radiation generated from the radioisotope-labeled fatty acid accumulated in the liver of the subject; and
  • the invention utilizes the fact that fatty acids accumulate in the liver where fatty acids are metabolized. More specifically, the invention can be carried out by administering radioisotope-labeled fatty acids as an imaging agent to the living body, detecting radioactivity generated from the liver where such imaging agents accumulate with a scintillation camera, etc., and noninvasively measuring the fatty acid metabolic behavior through the formation of an image (scintigraphy) based on the radiation attenuation.
  • fatty acids labeled with radioisotope radioisotope- labeled fatty acids
  • fatty acids in which iodine is replaced with radioactive iodine e.g., 123 I
  • radioactive iodine e.g., 123 I
  • fatty acids include 15- (p- iodophenyl)-9-methylpentadecanoic acid (9MPA) , 15- (p-iodophenyl) - pentadecanoic acid (IPPA), 15- (o-iodophenyl) -pentadecanoic acid (OPPA), 15- (p-iodophenyl) -3-methylpentadecanoic acid (BMIPP), 16- iodo-3-methylhexadecanoic acid (IMHA) , etc.
  • 123 I- BMIPP is preferable.
  • intravenous administration is preferable.
  • liver-targeted dynamic images are collected after a set period of time (usually after 5 minutes) . More specifically, such images are obtained as follows: bringing a scintillation camera close to the body surface area overlying the liver, collecting image data thereof, analyzing the collected image data, and thereby creating scintigram images.
  • Any scintillation camera can be selected without limitation according to the type of radioisotope for use in an administration agent.
  • a gamma camera and preferably, a gamma camera equipped with a low- energy parallel collimator can be mentioned.
  • time-radioactivity curve is created based on the created scintigram images.
  • the time-radioactivity curve shows attenuation, over time, of radiation generated from the liver, and the hepatic fatty acid metabolic behavior can be evaluated based on the time- radioactivity curve or the analytical results thereof.
  • hepatic fatty acid metabolic abilities of the subject before and after treatment can be easily compared, and thus the presence or absence of therapeutic effects (hypermetabolism) in the hepatic fatty acid metabolic disorder of the subject can be evaluated.
  • a method for objectively and quantitatively evaluating the hepatic fatty acid metabolic ability of the subject a method for calculating hepatic fatty acid clearance rate (%/min) can be mentioned.
  • the hepatic fatty acid clearance rate (%/min) can be calculated by the following steps of: (a) administering a radioisotope-labeled fatty acid to a subject;
  • the fatty acid clearance rate ( 123 I-BMIPP clearance rate) after therapeutic treatment is less than 0.5%/min, it ean be judged that no therapeutic effect is observed; and if the fatty acid clearance rate after therapeutic treatment is not less than 0.5%/min, it can be judged that a therapeutic effect is observed.
  • hepatic fatty acid ⁇ -oxidation ability of a subject can be evaluated based on the hepatic fatty acid clearance rate (%/min) .
  • %/min hepatic fatty acid clearance rate
  • the fatty acid clearance rate is less than 0.5%/min, it can be judged that the subject has a " lipid metabolism disorder and/or a risk of diseases caused thereby; and if the fatty acid clearance rate is not less than 0.5%/min, it can be judged that the subject has neither a lipid metabolism disorder nor a risk of diseases caused thereby.
  • the invention can achieve easy detection and judgment for the presence or absence of lipid metabolism disorder or a relative risk of a development disease caused by lipid metabolism disorder (lipid metabolism disorder-related diseases) with respect to each subject.
  • the detection method of the invention can be easily carried out in vitro while requiring no special knowledge, such as of a medical practitioner. With respect to a subject who is proved, by the detection method of the invention, to have relatively high potential risk a development of lipid metabolism disorder-related diseases, the judging result is notified to the subject, and then appropriate measures can be taken to prevent the development of lipid metabolism disorder- related diseases.. Therefore, the invention is extremely useful for preventing the onset and progression (advance) of lipid metabolism disorder-related diseases.
  • NASH patients or patients with a potential risk of NASH can be clarified as to whether or not the NASH is caused by lipid metabolism disorder (hepatic fatty acid ⁇ -oxidation ability disorder) .
  • lipid metabolism disorder hepatic fatty acid ⁇ -oxidation ability disorder
  • the invention provides a method for reducing the risk of developing lipid metabolism disorder-related diseases utilizing the lipid metabolism disorder susceptibility gene and the genetic polymorphism which participates in lipid metabolism disorder that are provided by the invention. Reduction of the risk is effective for preventing the onset of lipid metabolism disorder-related diseases and alleviating, if developed, such lipid metabolism disorder-related diseases.
  • the invention also provides a method for screening for an active ingredient effective for reducing the risk of developing lipid metabolism disorder-related diseases. According to the screening method of the invention, by obtaining such active ingredient, development of agents effective for reducing the risk of onset of lipid metabolism disorder-related diseases, and, in addition, preventing and/or treating lipid metabolism disorder-related diseases can be achieved.
  • the invention further provides a method for evaluating therapeutic effects of agents or other treatments on lipid metabolism disorder or lipid metabolism disorder-related diseases caused by having as a homozygote a genetic polymorphism that causes change (T ⁇ A) in the 94th amino acid of FABPl.
  • the method of the invention can noninvasively evaluate effects of administered agents and/or applied treatments, thereby appropriately treating patients and preventing mental and physical sufferings of patients caused by inappropriate treatments.
  • NASH non-alcoholic steatohepatitis
  • the patients were not being treated with methotrexate, corticoid, or insulin.
  • 123 I-BMIPP [ 123 I-labeled-15- (4-iodophenyl) -3 (R, S) - methylpentadecanoic acid] is an internally administered radiopharmaceutical (a cardiac disease diagnostic agent) used for diagnosis of the presence of cardiac diseases by evaluating local heart muscle fatty acid metabolism. After informed consent, the 37 patients (15 females and 22 males) were evaluated for their hepatic 123 I-BMIPP clearance ability by 123 I-BMIPP scintigraphy.
  • the patients fasted overnight and, received an intravenously administered 20 mL of physiological saline containing 111 Mbq 123 I-BMIPP, and lay in a bed equipped with a gamma (scintillation) camera.
  • the 123 I-BMIPP administered site was subjected to dynamic planner scanning (1 second x 60 frames, 30 lines x 60 frames) to determine the uptake of 123 I- BMIPP in the liver.
  • the gamma camera (GCA9300 A/HG, product of Toshiba Corporation) was used in combination with a low-energy high-resolution collimator (energy window: 159keV ⁇ 10%) .
  • Fig. 1 shows the results (images) obtained by measuring the amount of gamma rays (per 1 cm 2 for 30 seconds) in ROIs ((1) the heart, (2) the liver) with the lapse of time.
  • the regions indicated by (1) are ROIs of the heart, and the regions indicated by (2) are ROIs of the liver (S8 of the right hepatic lobe) .
  • the results show that 123 I- BMIPP moves to the liver within 1 minute after administration and the amount of 123 I-BMIPP in the liver peaks in about 5 minutes and then gradually declines.
  • Time-radioactivity curves (abscissa: time after administration of 123 I-BMIPP, ordinate: radioactivity) were made by plotting the amount of gamma rays (per 1 cm 2 for 30 seconds) in ROIs of the liver (S8 of the right hepatic lobe) measured using the gamma camera over a period of 1830 seconds at 30 second intervals from administration.
  • the time-radioactivity curves showed that 123 I-BMIPP moves to the liver within 60 seconds after administration and the amount of 123 I-BMIPP in the liver and peaks within 500 seconds after administration and decreases linearly over the following 1400 seconds.
  • the data in the region showing the linear decrease i.e., the data in the period from 510 seconds to 1830 seconds after administration of 123 I- BMIPP were used to make a maximum likelihood line according to the least sguares method.
  • the amount of 123 I-BMIPP at the time of 123 I-BMIPP administration was extrapolated, and this was set as 100% 123 I-BMIPP retention.
  • " 123 I-BMIPP clearance rate (%/min.)" indicating clearance of 123 I-BMIPP from the liver was calculated relative to the above extrapolated 123 I-BMIPP amount at the time of 123 I-BMIPP administration being set as 100% 123 I-BMIPP retention.
  • Fig. 2 shows the results showing hepatic 123 I-BMIPP retention (%) from immediately after to 1830 seconds after administration of 123 I-BMIPP (time-BMIPP retention curves) .
  • the results show that there are two types of groups in the NASH patients (37 persons) : a group showing a high clearance rate ( 123 I-BMIPP clearance rate: no less than 0.5 %/min. , indicated by white circles (O) in Fig. 2, 27 of the 37 patients) ; and a group showing a low clearance rate ( 123 I-BMIPP clearance rate: less than 0.5 % min., indicated by black circles (•) in Fig. 2, 10 of the 37 patients) .
  • genomic DNA was prepared according to the standard method. Using the thus prepared genomic DNA as a template, PCR (polymerase chain reaction) was performed at an annealing temperature of 59°C using primer 1 (AGTTGGAAGGTGACAATAAACTGGTGAAA) (SEQ ID: 11) and primer 2 (GTAGGAGGGTGGAGGGGTGGCATTAGGGTA) (SEQ ID: 12) to amplify the gene.
  • the genotype of the amplified gene was determined by the RFLP (restriction fragment length polymorphism) method using the restriction enzyme Hind III. More specifically, when the amplified gene was not cleaved by the restriction enzyme (Hind III) at all, the genotype was evaluated as A/A. When the gene was completely cleaved, the genotype was evaluated as G/G. When only half the amount of the amplified gene product was cleaved, the genotype was evaluated as A/G.
  • NASH patients Of the NASH patients (37 persons), those NASH patients having a polymorphism that causes a 94th amino acid change (T-»A) in FABPl, in the form of a homozygote (G/G) (hereinafter referred to as "FABPl variant NASH patients") received a 400 mg/day dosage of the fibrate drug "Bezafibrate" for 300 days.
  • FBPl variant NASH patients received a 400 mg/day dosage of the fibrate drug "Bezafibrate" for 300 days.
  • 123 I-BMIPP scintigraphy was performed to make time-radioactivity curves and time-BMIPP retention curves in the same manner as in (1) above and 123 I-BMIPP clearance rates (%/min) were determined.
  • Fig. 4 shows one example of the results. In Fig.
  • the graph indicated by black dots ( ⁇ ) is a time-BMIPP retention curve obtained by measurement before the administration of the fibrate drug
  • the graph indicated by white dots (O) is a time-BMIPP retention curve obtained by measurement after the administration of the fibrate drug.
  • the 123 I-BMIPP clearance rate (%/min) of all the FABPl variant NASH patients recovered from less than 0.5 %/min. (average of 0.216 %/min. in Fig. 4) to no less than 0.5 %/min. (average of 0.614 %/min in Fig. 4) upon the administration of the fibrate drug; and ameliorating effect (therapeutic effect) on NASH was obtained along with amelioration of fatty liver 8 to 10 months after beginning the administration of the fibrate drug.
  • Tamoxifen a potent antagonist of estrogen, is a pharmaceutical agent used for the treatment and prevention of breast cancer, but is known to cause non-alcoholic steatohepatitis (NASH) (tamoxifen-induced non-alcoholic steatohepatitis) in breast cancer patients.
  • NASH non-alcoholic steatohepatitis
  • tamoxifen-induced fatty liver Ten patients with tamoxifen-induced fatty liver, including 3 patients diagnosed as having tamoxifen-induced non ⁇ alcoholic steatohepatitis (tamoxifen-induced NASH) had their hepatic fatty acid beta-oxidation abilities measured using 123 I- BMIPP [ 123 I-labeled-15- (4-iodophenyl) -3 (R, S) -methylpentadecanoic acid] according to the method of Example 1 (1), and their genotype at the 3513rd position of the FABPl gene was examined according to the method of Example 1 (2) .
  • All the subject patients were such that their alcohol intake was not more than 2O g per day and they were not affected with any hepatic disease, other than non-alcoholic steatohepatitis (NASH) , such as viral hepatitis, hemochromatosis, Wilson's disease, or autoimmune liver diseases.
  • NASH non-alcoholic steatohepatitis
  • the patients were not being treated with methotrexate, corticoid, or insulin.
  • Table 1 shows the genotype at the 3513rd position of FABPl gene of the patients (a total of 10 persons) with tamoxifen-induced NASH or tamoxifen-induced fatty liver. [Table 1]
  • Tamoxifen has the action of inhibiting fatty acid beta- oxidation via antiestrogenic activity. Therefore, irrespective of the type of polymorphism at the 3513rd position of FABPl shown in
  • non-alcoholic steatohepatitis patients (15 females, 22 males, a total of 37 persons) used as subjects in Example 1 and the tamoxifen-induced NASH patients (3 females) used as subjects in Example 2, a total of 40 persons, were further subjected to the following tests.
  • Optical microscopic examination (measurement of fat occupying area of the hepatic lobule)
  • the liver biopsy specimens obtained from the patients were fixed in 10% phosphate buffered formalin (pH 7.4) according to the standard method and embedded in paraffin to produce cell blocks, followed by staining with hematoxylin and eosin.
  • the lipid droplets area (fat occupying area) of the hepatic lobule was calculated using NIH (National Institutes of Health) software "image 16.2" (http://rsb.info.nih.gov/nih-image/).
  • Computed tomography was performed on all the patients.
  • CT images were obtained as cross-sectional slices with a sectional thickness of 10 mm at 10 mm intervals and fixed at WL 30 and WW 300 using helical-CT (ProSeed, General Electric Yokogawa Medical Systems) .
  • Regions of interest (RIO) with a diameter of 10 mm in eight hepatic couinaud segments and one splenic segment were subjected to non-enhanced scan while avoiding blood vessels to obtain CT attenuation values.
  • the liver/spleen ratio was calculated using the minimum CT value of the liver.
  • the visceral fat occupying area and subcutaneous fat occupying area were determined at the umbilical level using NIH images.
  • BMI body mass index
  • tamoxifen a potent antagonist of estrogen
  • NASH non-alcoholic steatohepatitis
  • fibrate drugs e.g., Bezafibrate
  • PPAR- ⁇ peroxisome proliferator-activated receptor- ⁇
  • Example 2 show that all the tamoxifen- induced NASH patients had reduced hepatic fatty acid beta- oxidation abilities ( 123 I-BMIPP clearance rate: less than 0.5%/min.). Therefore, after informed consent, a fibrate drug (Bezafibrate) (400 mg/day) was administered to the tamoxifen- induced NASH patients (3 persons) over a period of 1 year. Fat accumulation in the liver and 123 I-BMIPP clearance rate determined from CT (computed tomography) and 123 I-BMIPP scintigraphy, respectively, after 1 year of administration were compared to those determined before the administration to evaluate therapeutic effects of the fibrate drug.
  • Bezafibrate 400 mg/day
  • the non-alcoholic steatohepatitis (NASH) patients (37 persons) were classified into the following 4 groups, based on the 123 I-BMIPP clearance rates determined in Example 1 and the body mass index (BMI) determined in (3) above (Fig. 5) .
  • a 123 I- BMIPP clearance rate of at least 0.5%/min. is defined as a "high 123 I-BMIPP clearance rate", and a 123 I-BMIPP clearance rate of less than 0.5%/min. as a "low 123 I-BMIPP clearance rate”.
  • Patients with a body mass index (BMI) of at least 30 were defined as obese patients, and those with a body mass index (BMI) of less than 30 as non-obese patients.
  • Group A Obese NASH patients with a high 123 I-BMIPP clearance rate (12 persons)
  • Group B Non-obese NASH patients with a high 123 I-BMIPP clearance rate (15 persons) >
  • Group C Non-obese NASH patients with a low 123 I-BMIPP clearance rate (9 persons)
  • Group D Obese NASH patients with a low 123 I-BMIPP clearance rate (1 person)
  • Table 2 shows the summary of clinical characteristics of the above groups A to C. [Table 2]
  • group A consisting of obese NASH patients showed significant accumulations of subcutaneous fat (p ⁇ 0.01) and visceral fat (p ⁇ 0.01) (both groups had a high 123 I- BMIPP clearance rate) .
  • these two groups had many metabolic syndrome features, including visceral obesity, fatty liver, and a HOMA-IR (homeostasis model of assessment of insulin resistance) increase with high fasting insulin.
  • Group C consisting of non-obese NASH patients (low 123 I-BMIPP clearance rates) had a visceral fat occupying area of 138.7 ⁇ 51.8 cm 2 , a liver biopsy specimen fat occupying area of 67.8 ⁇ 12.0 %, and a HOMA-IR (homeostasis model of assessment of insulin resistance) of 2.71 ⁇ 2.2 cm 2 , i.e., all high values, but group C had a BMI of 25.4 ⁇ 2.9 kg/m 2 , i.e., a low value, unlike group A consisting of obese NASH patients.
  • FIG. 6 shows the relationships between the body mass index (BMI) (kg/m 2 ) and fat occupying area (%) obtained from liver biopsy specimens from the 37 NASH patients.
  • ⁇ indicates the results of group C patients (non-obese NASH patients with a low 123 I-BMIPP clearance rate ( ⁇ 0.5 %/min.)). All the patients in this group had a liver biopsy specimen fat occupying area of more than 50% . However, no significant differences were observed between the 3 groups in terms of hepatic inflammation and fibrosis.
  • beta-oxidation promoters e.g., fibrate drugs
  • Fig. 7 shows the results of measuring liver fat accumulation in 3 tamoxifen-induced NASH patients by CT before and after 1 year of Bezafibrate administration.
  • FIG. 7 treatment with Benzafibrate effectively reduced liver fat accumulation.
  • the upper diagram shows the result before Bezafibrate administration, and the lower diagram shows the result after 1 year of Bezafibrate administration.
  • the patients 3 persons
  • their 123 I-BMIPP clearance rates were remarkably increased (0.625%/min. , 0.917%/min., and
  • Fig. 8 shows a representative result. Black circles show the results before Bezafibrate administration, and open circles show the results after 1 year of Bezafibrate administration. These results are in agreement with a previously published article (Saibara T, Onishi S, Ogawa Y, Yoshida S, Enzan H, Bezafibrate for tamoxifen-induced nonalcoholic steatohepatitis, Lancet. 999; 353 (9166) : 1802) . These results also suggest that therapeutic effects of various pharmaceuticals and therapies on NASH patients can be evaluated in a reproducible and semi-quantitative manner by evaluating the 123 I-BMIPP clearance rate using 123 I-BMIPP scintigraphy.
  • hepatic 123 I-BMIPP clearance was investigated using 123 I-BMIPP scintigraphy and the obtained time-radioactivity curves confirmed that 123 I-BMIPP was uptaken by the liver within 1 minute after administration of 123 I- BMIPP.
  • the 123 I-BMIPP concentration in the liver peaked within 500 seconds after administration of 123 I-BMIPP and then linearly declined over the following 1400 seconds.
  • the experimental data were reproduced by repeated measurements performed 3 months later. This shows that hepatic 123 I-BMIPP clearance rates can be analyzed in a reproducible manner using 123 I-BMIPP scintigraphy. Since the evaluation of internal fatty acid beta-oxidation ability using 123 I-BMIPP can be used to measure the reduction of hepatic fatty acid metabolism in a quantitative and reproducible manner and is also clinically very easy, it can be used easily by general doctors .
  • Imaging diagnostic methods such as computed tomography (CT) , ultrasonography (US) , and magnetic resonance imaging methods (MRI) merely visualize the presence of fatty infiltration in hepatic parenchyma and cannot show dynamic functions of the liver.
  • CT computed tomography
  • US ultrasonography
  • MRI magnetic resonance imaging methods
  • the above method can illustrate fatty acid metabolic capability and enable NASH patients to select a suitable treatment.
  • Fatty acid analyses based on similar principles, such as PET (positron emission tomography) and positron CT, would be easily performed if suitable labeling agents were to be developed.
  • Example 4 Ninety-two NASH patients and 198 healthy persons were examined for their genotype at the 3513rd position of the FABPl gene according to the method of Example 1 (2) and the frequency of appearance of homozygous guanine (G/G) at the 3513rd position of the FABPl gene and the frequency of possession of the variant FABPl gene (a3513g) were compared.

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Abstract

La présente invention concerne un technique de détection et d'appréciation de troubles du métabolisme lipidique et des risques que présente le sujet de contracter des maladies qui en découlent, ainsi que des réactifs (sonde et amorce par exemple) pour le diagnostic desdits troubles, et un trousse de réactifs comprenant au moins un tel réactif, qui tous peuvent être utilisés efficacement avec la technique de détection de l'invention. La présente invention permet de détecter un polymorphisme génétique entraînant un changement (TRA) du 94me acide aminé (protéine de liaison 1 des acides gras, foie [Homo sapiens] chez un sujet.
PCT/JP2006/305909 2005-04-28 2006-03-17 Technique de detection de troubles du metabolisme des lipides et agent diagnostique utilise avec ces technique Ceased WO2006117945A1 (fr)

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EP2157431A1 (fr) 2008-08-11 2010-02-24 One Way Liver Genomics, S.L. Procédé pour le diagnostic de la stéato-hépatite non alcoolique utilisant des profils métaboliques
WO2011036117A1 (fr) 2009-09-22 2011-03-31 One Way Liver Genomics, S.L. Méthode diagnostique de la stéatose hépatique non alcoolique basée sur le profil métabolique
CN119144433A (zh) * 2024-09-25 2024-12-17 陕西中医药大学 一种叶酸代谢相关基因型检测装置及其检测方法

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JP6272065B2 (ja) * 2014-02-07 2018-01-31 国立研究開発法人国立国際医療研究センター Ｎａｓｈ診断プログラム、ｎａｓｈ診断装置の作動方法、及びｎａｓｈ診断装置
JP6494356B2 (ja) * 2015-03-24 2019-04-03 国立大学法人旭川医科大学 非アルコール性脂肪性肝疾患及び／又は非アルコール性脂肪肝炎の発症リスク及び／又は重症化リスクの判定方法、並びに該判定用オリゴヌクレオチドキット
AU2020320244B2 (en) * 2019-07-30 2024-10-10 Kobiolabs, Inc. Composition and method for preventing, alleviating, or treating liver injury

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2157431A1 (fr) 2008-08-11 2010-02-24 One Way Liver Genomics, S.L. Procédé pour le diagnostic de la stéato-hépatite non alcoolique utilisant des profils métaboliques
US8563318B2 (en) 2008-08-11 2013-10-22 One Way Liver Genomics, S.L. Method for the diagnosis of non-alcoholic steatohepatitis based on a metabolomic profile
WO2011036117A1 (fr) 2009-09-22 2011-03-31 One Way Liver Genomics, S.L. Méthode diagnostique de la stéatose hépatique non alcoolique basée sur le profil métabolique
CN119144433A (zh) * 2024-09-25 2024-12-17 陕西中医药大学 一种叶酸代谢相关基因型检测装置及其检测方法

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