WO2006117945A1

WO2006117945A1 - Method for detecting lipid metabolism disorder, and diagnostic agent for use therein

Info

Publication number: WO2006117945A1
Application number: PCT/JP2006/305909
Authority: WO
Inventors: Toshiji Saibara
Original assignee: Kochi University
Priority date: 2005-04-28
Filing date: 2006-03-17
Publication date: 2006-11-09
Also published as: JP2008538893A

Abstract

The present invention provides a method for detecting and judging lipid metabolism disorders and a risk of developing diseases caused thereby, and 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 can be accomplished by detecting a genetic polymorphism that causes a change (TRA) of the 94th amino acid of FABP1 (fatty acid binding protein 1, liver [Homo sapience]) in a subject.

Description

DESCRIPTION

METHOD FOR DETECTING LIPID METABOLISM DISORDER, AND

DIAGNOSTIC AGENT FOR USE THEREIN

TECHNICAL FIELD

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.

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.

BACKGROUND ART

Metabolic syndrome (metabolic disorder 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.

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.

Recently, in addition to hypertension, diabetes and hyperlipidemia, non-alcoholic steatohepatitis (hereinafter also referred to as "NASH") has been attracting attention as a new lifestyle-related disease. NASH is a 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. However, since diagnosis of NASH is difficult and liver biopsy is necessary for clear diagnosis, many cases are currently overlooked. Further, treatment of fatty liver varies depending on the cause: fatty liver due to obesity can be treated by weight reduction through dietary measures and the like, while fatty liver due to alcohol toxicity can be treated by abstinence from alcohol. That is, fatty liver can be treated by eliminating the cause. However, the cause of NASH has not yet been clarified, and therefore no effective therapy for the disease has been established. With recent rapid progress in genome analysis technology, associations between diseases and genes are gradually being clarified. For example, attention is focused on genetic polymorphisms [for example, SNPs (single nucleotide polymorphisms)], as genetic changes that can cause diseases. Researchers around the world are actively studying associations between genetic polymorphisms and diseases.

In the human genome, 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.

In view of recent concerns about the increasing incidence of lifestyle-related diseases and metabolic syndrome accompanying the increasing incidence of obesity, to improve the prognosis of lifestyle-related diseases, it is especially important to establish a method for identifying individuals that are highly susceptible to lifestyle-related diseases including NASH, and to treat such diseases at an early stage. The following are documents related to the present invention .

[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 ¹²³I-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., Εffect 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

DISCLOSURE OF THE INVENTION

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.

Further, the inventors performed tests on patients with non-alcoholic steatohepatitis (NASH) , and found that fatty acid beta-oxidation in the liver is compensatorily enhanced in the majority of cases, while in the remaining cases, no compensatory enhancement of fatty acid beta-oxidation in the liver occurs and fatty acids are not metabolized. The inventors also found that the latter NASH patients with insufficient fatty acid metabolism generally have the above-mentioned homozygotic genetic polymorphism. Specifically, the inventors found that 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.

Further, the inventors confirmed that this genetic polymorphism (genetic abnormality) 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.

Furthermore, 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.

Based on the above findings, 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.

(1) 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) ] .

(1-2). The method according to (1-1), which is a method for detecting a genetic polymorphism existing at locus 2pll on human chromosome 2.

(1-3). The method according to (1-1) or (1-2), wherein the genetic polymorphism is a homozygotic genetic polymorphism in the FABPl gene.

(1-4) . 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.

(1-7) . A method for detecting a lipid metabolism disorder or a risk of disease caused thereby, comprising the following steps (a) to (c) :

(a) extracting genomic DNA from a biological sample of a subject;

(b) detecting the 3513rd base of the base sequence of the FABPl gene contained in the extracted genomic DNA; and

(c) identifying whether the base is A or G.

(1-8) The method according to (1-7), further comprising the following step (d) :

(d) if the base is homozygous for G (G/G) , determining that the subject has a lipid metabolism disorder or a risk of a disease caused thereby.

(1-9) . 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;

(ii) calculating a fatty acid clearance rate (%/min) in the liver from the change in the radioactivity detected over time in step (i) ; and

(iii) identifying whether the fatty acid clearance rate is less than 0.5%/min or not less than 0.5%/min.

(1-10) 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.

(1-11) . 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.

(1-12) . 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. (1-13) . 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.

(1-14) . 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.

(1-15) . 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.

(1-17). The probe according to (1-15) or (1-16), which is immobilized on a solid phase. (1-18) . 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. (1-19). The reagent or reagent kit according to (1-18), wherein the lipid metabolism disorder or disease caused thereby is a disorder of hepatic fatty acid beta-oxidation ability or disease caused thereby.

(2) Method for detecting NASH of hepatic fatty acid beta- oxidation disorder-type, and reagent for use therein

Diseases caused by insufficient hepatic fatty acid beta-oxidation ability include non-alcoholic steatohepatitis (NASH) as indicated in (1-6) . As mentioned above, 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) . When people of fatty acid beta-oxidation ability disorder-type continue to eat high-fat diets, they are unable to compensatorily enhance fatty acid beta- oxidation in the liver, and thus are likely to develop serious fatty liver and have a high risk of developing non-alcoholic steatohepatitis (NASH) . To reduce such a risk, artificial or forcible induction of fatty acid beta-oxidation in the liver is effective. For NASH of normal hepatic fatty acid beta-oxidation ability-type, a different therapeutic method needs to be considered since the causes of the disease are different from those of NASH of hepatic fatty acid beta-oxidation ability disorder-type. It is therefore clinically necessary to differentiate, in NASH patients, whether the NASH is of normal hepatic fatty acid beta-oxidation ability-type or of hepatic fatty acid beta-oxidation ability disorder-type. Thus, as shown below, 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.

(2-1) . 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

FABPl .

(2-2). The method according to (2-1), which is a method for detecting a genetic polymorphism existing at locus 2pll on human chromosome 2. (2-3). The method according to (2-1) or (2-2), wherein the genetic polymorphism is a homozygotic genetic polymorphism in the FABPl gene.

(2-4) . The method according to any one of (2-1) to (2-

3) , which is a method for detecting the presence or absence of the genetic polymorphism using change (T→A) of the 94th amino acid of FABPl as an index.

(2-5) . 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;

(c) identifying whether the base is A or G. (2-6). The method according to (2-5), further comprising the following step (d) :

(d) if the base is homozygous for G (G/G) , determining that the NASH is of hepatic fatty acid beta-oxidation ability disorder-type. _t (2-7) . 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) :

(i) administering a radioisotope-labeled fatty acid to the subject and detecting, over time, radioactivity generated 5. from the radioisotope-labeled fatty acid accumulated in the liver;

(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.

(2-8). The method according to (2-7), further comprising the following step (iv) :

(iv) if the fatty acid clearance rate is less than 5 0.5%/min, determining that the NASH is of hepatic fatty acid beta-oxidation ability disorder-type; and if the fatty acid clearance rate is not less than 0.5%/min, determining that the NASH is of normal hepatic fatty acid beta-oxidation-type.

(2-9) . 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 .

(2-10) . 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. 5 (2-11) . 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 nucleotide of the base sequence .

(2-12) . 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.

(2-14) . A reagent for detecting NASH of hepatic fatty acid beta-oxidation ability disorder-type, the reagent 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 .

(3) Method for reducing the risk of developing lipid metabolism disorder-related diseases (3-1) . 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) .

(3-2). The method according to (3-1), wherein the lipid metabolism disorder-related disease is NASH of hepatic fatty acid beta-oxidation ability disorder-type.

(3-3) . A method of reducing the risk of developing NASH, and in particular NASH of hepatic fatty acid beta-oxidation ability disorder-type, in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G) , comprising administering a fibrate drug to the subject.

(3-4) . A method for preventing the development of NASH, and in particular NASH of hepatic fatty acid beta-oxidation ability disorder-type, in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G) , comprising administering a fibrate drug to the subject.

(3-5) . A method for alleviating NASH, and in particular NASH of hepatic fatty acid beta-oxidation ability disorder-type, in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G), comprising a fibrate drug to the subject.

(3-6) . 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.

(4) Method for screening for components that are effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related diseases

(4-1) . A method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease, the method 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;

(b) measuring the expression level of FABPl or variant FABPl, or FABPl activity of 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.

(4-2) . A method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease, the method comprising the following steps of:

(i) administering a test substance to a cell comprising polynucleotide which is a FABPl gene on human chromosome 2 or a segment of such a gene and at least has base sequences represented by SEQ ID No. 2 and SEQ ID No. 3 on the 5' and 3' sides, respectively, of each side of a base Guanine (G) ; and

(ii) selecting, as a candidate substance from the administered test substances, a substance for inducing transition to a base Adenine (A) from the base Guanine (G) placed between the base sequences represented by SEQ ID No. 2 and SEQ ID No.3 of the polynucleotide of the cell.

(4-3) . The screening method according to (4-1) or (4- 2) , wherein the lipid metabolism disorder or disease caused thereby is a hepatic fatty acid β-oxidation disorder or disease caused by such disorder.

(5) Method for evaluating therapeutic effects or disease risk reducing effects on lipid metabolism disorders or lipid metabolism disorder-related diseases (5-1) . 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, wherein hepatic fatty acid metabolic abilities are compared before and after the treatment.

(5-2) . The method according to (5-1) , wherein the hepatic fatty acid metabolic ability is measured by a method comprising the following steps (a) and (b) : (a) administering a radioisotope-labeled fatty acid to a subject, and detecting, over time, radiation generated from the radioisotope-labeled fatty acid accumulated in the liver; and

(b) determining hepatic fatty acid metabolic ability based on the change in the radiation detected over time in step (a).

(5-3) . 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, the method comprising the following steps of:

(i) administering a radioisotope-labeled fatty acid to a subject, and detecting, over time, radiation generated from the radioisotope-labeled fatty acid accumulated in the liver;

(ii) calculating a fatty acid clearance rate (%/min) in the liver based on the change in the radiation detected over time in step (i) ; and

(iii) if the fatty acid clearance rate is less than 0.5%/min, determining that the treatment has a therapeutic effect or a disease risk reducing effect; and if the fatty acid clearance rate is not less than 0.5%/min, determining that the treatment has neither therapeutic effect nor a disease risk reducing effect.

BRIEF DESCRIPTION OF DRAWING

Fig. 1 shows the results of ¹²³I-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 ¹²³I-BMIPP retention (%) in non-alcoholic steatohepatitis (NASH) patients (37 persons) from immediately after to 1830 seconds after the administration of ¹²³I-BMIPP (Example 1 (I)). The patients are clearly divided into two groups: a group of patients showing a ¹²³I-BMIPP clearance rate of less than 0.5 % min (average of 0.27 %/min) (Φ) , and a group of patients showing a ¹²³I-BMIPP clearance rate of at least 0.5 %/min. (average of 0.79 %/min.) (O) .

Fig. 3 shows the relationship between the ¹²³I-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)). In Fig. 3, "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; and "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 (φ) 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 ¹²³I-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) and fat occupying area (%) obtained from 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 (φ) and after (O) 1 year of Bezafibrate administration (Example 3) . BEST MODE FOR CARRYING OUT THE INVENTION

1. Definition of terms used in the present invention In this specification and the appended claims, abbreviations for base sequences (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.

In this specification and the appended claims, unless otherwise stated, 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", "oligonucleotide" and "polynucleotide" have 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. Specifically, in a population of humans, when one or more individuals have a variation from another individual, such as substitution, deletion, insertion* transition, inversion and/or the like, of one or more nucleotides at specific sites of their genomes, and when it is statistically evident that such a variation is not a mutation in the one or more individuals or it is genealogically demonstrated that such a variation is not a mutation in the one or more individuals and occurs at a frequency of 1% or more, the variation is referred to as a "genetic polymorphism". "Genetic polymorphism" as used herein 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.

As used herein, "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.

Diseases caused by such lipid metabolism disorders, and in particular fatty acid beta-oxidation ability disorders in the liver, include hepatic fatty acid beta-oxidation disorder, nonalcoholic steatohepatitis (NASH) , fatty liver and myocardial infarction. "Non-alcoholic steatohepatitis (NASH)" encompasses tamoxifen-induced non-alcoholic steatohepatitis and other types of non-alcoholic steatohepatitis, and "fatty liver" encompasses non-alcoholic fatty liver and non-obese fatty liver.

Further, in this specification, "gene frequency" refers to the frequency of one allele among all alleles at a gene locus in a population.

As used herein, "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. Thus, 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. For example, 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. As used herein, "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.

2. Lipid metabolism disorder susceptibility gene The 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

Brouillette C, Bosse Y, Perusse L, Gaudet D, Vohl MC, "Effect of liver fatty acid binding protein (FABP) T94 A missense mutation on plasma lipoprotein responsiveness to treatment with fenofibrate", J. Hum. Genet. 2004; 49 (8): 424-32, Epub 2004 JuI. 13) .

The present inventors found through their research activities that patients with non-alcoholic steatohepatitis (NASH) 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.

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. In NASH patients of hepatic fatty acid beta- oxidation disorder-type, 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") .

Further, 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.

Further, 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. In view of this, 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.

Thus, it can be considered that detection of the FABPl change (T94A) , or a genetic abnormality or genetic polymorphism that brings about such an amino acid change makes it possible to diagnose, in individual subjects, a lipid metabolism disorder due to insufficient hepatic fatty acid beta-oxidation ability, and a risk of diseases caused by the disorder. Based on the above, 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. Among such genes, 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

(mitochondrial ribosomal protein L45 pseudogene 1) , all of which are close to the FABPl 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, mitochondrial (mitofilin) MRPL35 : mitochondrial ribosomal protein L35 C2orf23 : chromosome 2 open reading frame 23 JMJDlA : jumonji domain containing IA

VPS24 : vacuolar protein sorting 24 (yeast) RNFl03 : ring finger protein 103 FLJ13910 : hypothetical protein FLJ13910 CD8A : CD8 antigen, alpha polypeptide (p32) 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 L0C51315 SMYDl : SET and MYND domain containing 1 FLJ10916 : hypothetical protein FLJ10916

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 variable 3-34

3. 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. Preferable examples of 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. Examples of diseases caused by such lipid metabolism disorders (lipid metabolism disorder-related diseases) , and in particular hepatic fatty acid beta-oxidation ability disorders, 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) .

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) . Examples of such 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. As used herein, "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.

Examples of such 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. Specific examples include 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) ] .

Further, in addition to genes (and oligo- and polynucleotides) containing genetic predisposition markers, gene products (e.g., mRNA, its derivative (cDNA) , proteins) reflecting the genetic predisposition can be used as diagnostic markers for lipid metabolism disorders and/or diseases caused thereby.

Examples of 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.

4. Method for detecting lipid metabolism disorder or risk of disease caused thereby

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) . In genetics, 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. However, "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.

Specifically, 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.

As a more specific example, 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) :

(a) extracting genomic DNA from a biological sample of a subject, and ► (b) detecting the 3513rd base of the base sequence of the FABPl gene in the extracted genomic DNA.

In actual operation of 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.

When such detection detects that the SNP 3513 base is homozygous for G (guanine) (G/G) , 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) . When such detection detects that 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) .

It is preferable that the method of the present invention further comprises the following steps (c) and (d) :

(c) identifying whether the 3513rd base of the base sequence of the FABPl gene (i.e., the base between the base sequences shown in SEQ ID NOS: 2 and 3) is A or G,

(d) if the base is homozygous for G (G/G) , determining ■that the subject has a lipid metabolism disorder and is likely to develop a lipid metabolism disorder-related disease (or have a risk of a lipid metabolism disorder-related disease) .

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. According to the present invention, 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. Thus, 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 above steps (a) (extraction of genomic DNA) and (b) (detection of the target base) can be performed by known methods [e.g., Bruce, et al., Genome Analysis/A Laboratory Manual (Vol. 4), Cold Spring Harbor Laboratory, NY (1999)].

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.

For example, 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. al., "Molecular Cloning: A Laboratory Manual (2nd Ed.)", Cold Spring Harbor Laboratory, NY) , a method using a commercially available DNA extraction kit, or the like. Further, if necessary, DNA containing the FABPl gene on human chromosome 2 or exon 2 thereof may be isolated. Such 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. In 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) .

In addition to direct sequencing of the corresponding region of the gene as described above, 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 polymorphism- specific oligoprobes, and the genotype is determined by using temperature-dependent differences in that the probes form a secondary structure in its molecule or hybridize with the target region, etc. Also usable are a method in which a base extension reaction of template-specific primers is performed using polymerases, to identify bases incorporated into the polymorphic regions by the reaction (e.g., a method in which fluorescence from fluorescently labeled dideoxynucleotides is detected, a method in which the incorporated dideoxynucleotides are detected by mass spectrometry) , and a method in which, subsequent to the use of template-specific primers, the presence or absence of a complementary or non-complementary base pair is recognized by an enzyme) .

Typical known methods for detecting genetic polymorphisms are listed below, but are not intended to limit the scope of the present invention.

(a) RFLP (restriction enzyme fragment length polymorphism) methods, (b) PCR-SSCP (single-stranded DNA conformation polymorphism analysis) methods [Biotechniques, 16, 296-297 (1994) and Biotechniques, 21, 510-514 (1996)], (c) ASO (allele specific oligonucleotide) hybridization [Clin. Chim. Acta, 189, 153-157 (1990)], (d) direct sequencing [Biotechniques, 11, 246-249 (1991)], (e) ARMS (Amplification Refracting Mutation System) methods [Nuc. Acids. Res., 19, 3561-3567 (1991); Nuc. Acids. Res., 20, 4831-4837 (1992)], <f) DGGE (Denaturing Gradient Gel Electrophoresis) [Biotechniques, 27, 1016-1018 (1999)], (g) RNase A cleavage [DNA CeIl. Biol., 14, 87-94 (1995)], (h) chemical cleavage [Biotechniques, 21, 216-218 (1996)], (i) DOL (Dye-labeled Oligonucleotide Ligation) methods [Genome Res., 8, 549-556 (1998)], (j) TaqMan-PCR [Genet. Anal., 14, 143-149 (1999); J. Clin. Microbiol., 34, 2933-2936 (1996)], (Jc) invader methods [Science, 5109, 778-783 (1993); J. Biol. Chem. , 30, 21387-21394 (1999); Nat. Biotechnol., 17, 292-296 (1999)], (1) MALDI-TOF/MS (Matrix Assisted Laser Desorption Time-of- Flight/Mass Spectrometry) [Genome Res., 7, 378-388 (1997); Eur. J. Clin. Chem. Clin. Biochem. , 35, 545-548 (1997)], (m) TDI

(Template-directed Dye-terminator Incorporation) [Proc. Natl. Acad. Sci. USA, 94, 10756-10761 (1997)], (n) molecular beacon method [Nat. Biotechnol., 1, 49-53 (1998); Gene & Medicine, 4, 46-48 (2000)], (o) DASH (Dynamic Allele-Specific Hybridization) [Nat. Biotechnol., 1, 87-88 (1999); Gene & Medicine, 4, 47-48 (2000)], (p) padlock probe method [Nat. Genet., 3, 225-232 (1998); Gene & Medicine, 4, 50-51 (2000)], (q) UCAN method [website of TAKARA SHUZO CO., LTD. (http://www.takara.co.jp) ], (r) DNA chips and DNA microarrays ("SNP Idenshi Takei no Senryaku (SNP genetic polymorphism strategy)", Kenichi Matsubara and

Yoshiyuki Sakaki, Nakayama Shoten, 128-135) , and (s) ECA method [Anal. Chem., 72, 1334-1341 (2000)].

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) . If the method of the present invention detects in a subject a lipid metabolism disorder and a relatively high risk of developing a disease caused by the disorder, it is possible to inform the subject of the fact and take appropriate measures to prevent development of lipid metabolism disorder-related diseases. Therefore, the method of the present invention is a very useful for preventing development of lipid metabolism disorder-related diseases and any further advance (progress) of such diseases.

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. Examples of 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.

5. Method for detecting NASH of hepatic acid beta-oxidation disorder-type

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. This discernment is based on the fact that there are two types of NASH patients: those having a lipid metabolism disorder due to an inherent homozygotic genetic polymorphism that causes a 94th amino acid change (T-»A) in FABPl (hepatic fatty acid beta-oxidation ability disorder-type) , and those having no lipid metabolism disorder [normal lipid metabolism-type (normal hepatic fatty acid beta-oxidation-type) ] . 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

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. As a result of the detection, 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) . If a NASH patient is deemed to be of lipid metabolism disorder-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. Further, when a subject with a potential risk of NASH is deemed to be of lipid metabolism disorder-type, 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) .

Accordingly, such 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) .

The below described reagents (probe, primer, labeled probe and labeled primer) for detecting a lipid metabolism disorder or a susceptibility to (a risk of developing of) a lipid metabolism disorder-related disease, and kit comprising such reagents, can also be used as reagents (probe, primer, labeled probe and labeled primer) 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.

6. Reagent for detecting lipid metabolism disorder or risk of lipid metabolism disorder-related disease, and a kit comprising the reagent

(1) Probe

To detect the target genetic polymorphism (a lipid metabolism disorder susceptibility SNP, for example SNP3513) and nucleotides containing the base, 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.

As used herein, "specifically hybridize" means that such an 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.

Examples of such 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. Specifically, 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.

More preferably, as described hereinafter, 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 (oligo- or polynucleotide) may also be used immobilized on a solid phase. Thus, 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. With respect to .the immobilization method, for example, a commercially available spotter (e.g., a product of Amersham) can be used to obtain a DNA microarray. 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

(Affymetrix) or inkjet printing technique (Rosetta Inpharmatics) ] .

When employing the TaqMan PCR method [Livak KJ. , Gene Anal. 14, 143 (1999), and Morris T et al., J Clin Microbiol 34, 2933 (1996)], which is an example of an ASO method, 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.

In the invader method [Lyamichev V. et al., Nat Biotechnol 17, 292 (1999)], which is another example of an ASO method, 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.

(2) Primer

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. Such a primer (oligonucleotide) 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.

Examples of such 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

3513 site in the FABPl gene to thereby specifically amplify the continuous oligo- or polynucleotide.

Examples of such 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. Specific examples include an 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.

A specific method of using the primer is described below, in which MALDI-TOF/MS (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry) is applied to the Mass Array method [Haff LA et al., Genome Res. 7, 378 (1997); Little DP et al., Nature Medicine Vol. 3, No. 12, 1413-1416

(1997)]. In this case, 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. In this method, the primer usually consists of at least 15 bases, and is preferably designed to be as short as possible.

(3) Labeled probe and primer 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. ► Fluorochromes preferably usable in the present invention include those generally used for labeling nucleotides for detection or quantitation of nucleic acids. Examples of such flurochromes 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)].

Commercially available 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. Examples of such 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. When the TaqMan-PCR method is used in the genetic polymorphism detecting method, which is one embodiment of the present invention, 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 used as reagents for detecting a lipid metabolism disorder or susceptibility to or a risk of a disease caused thereby.

(4) A reagent kit for detecting lipid metabolism disorder or risk of a lipid metabolism disorder-related disease

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.

7. Method for reducing risk of lipid metabolism disorder-related diseases As described above, 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 β-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.

Moreover, 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.

Based on the above findings, 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. In addition, in accordance with the same idea as above, 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") .

There is no limitation to the specific techniques for practicing these methods insofar as the above-described objects can be achieved, and, for example, well-known processes as well as techniques to be developed in the future can be employed. Moreover, instead of the above-described methods, administration of normal FABPl or normal FABPl gene also can treat lipid metabolism disorder and also reduce the risk of developing lipid metabolism disorder-related diseases. In view of this point, 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.

Still further, instead of the above-described methods, artificial or forcible compensation for hepatic fatty acid β- oxidation ability (e.g., administration of an agent for increasing fatty acid β-oxidation ability, etc.) also can treat lipid metabolism disorder and also can reduce the risk of developing lipid metabolism disorder-related diseases. Examples of agents for increasing fatty acid β-oxidation ability include fibrate drugs (e.g., clofibrate, aluminium clofibrate, simfibrate, clinofibrate, bezafibrate) as described above.

8. Method for screening for components that are effective for reducing risk of lipid metabolism disorder-related diseases 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) .

(8-1) The screening can be carried out by the following steps of:

(a) contacting a test substance with a test cell expressing variant FABPl [FABPl (T94A) ] in which the 94th amino acid residue is alanine;

(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.

The 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.]. Among the above, 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.

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. When 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.

In this method, subsequently, 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. Moreover, 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. Various 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.

(8-2) The screening method of the invention can be carried out by the following steps of:

(1) administering a test substance to a cell comprising polynucleotide which is a FABPl gene on human chromosome 2 or a segment of such a gene and at least has base sequences represented by SEQ ID No. 2 and SEQ ID No. 3 on the 5' and 3¹ sides, respectively, of each side of a base Guanin (g) ; and

(2) selecting, as a candidate substance from the administered test substances, a substance for inducing transition to a base Adenine (A) from the base Guanine (G) placed between the base sequences represented by SEQ ID No. 2 and SEQ ID No.3 of the polynucleotide of the cell.

Step (2) (step of selecting a candidate substance) 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. Nor is 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. can be carried out by a suitably-selected method well known in this field [e.g., Maniatis, T.et al., "Molecular Cloning-A Laboratory Manual", Cold Spring Harbor Laboratry, NY, (1982); WO2002-52000; "New biochemical experiment lecture 18, Cell-culture engineering" edited by Japanese Biochemical Society, Tokyo Kagaku Dojin, (1990), 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.

(8-3) The screening method of the invention can be carried out by the following steps of:

(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.

Substances that bind in vivo to FABPl can be mentioned as examples of 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. Subsequently, the genetic change human FABPl is purified and obtained using a purification column. There is no limitation to the method for measuring binding affinity between variant FABPl and a FABPl-binding substance. For example, the binding affinity therebetween can be measured based on the binding reaction of a labeled oleic acid, or the like, which is known to bind to FABPl, etc. Alternatively, 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. For example, 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. Note that 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. 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) .

9. Method for evaluating therapeutic effects or disease risk reducing effects

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 β- 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 β-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.].

Considering the above, by artificially or inducibly enhancing hepatic fatty acid metabolism, more specifically, hepatic fatty acid β-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) . This can prevent the onset of diseases caused by 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.) and, if developed, can alleviate them.

In other words, such therapeutic effects can be evaluated by comparing hepatic fatty acid metabolic abilities before and after treatment, more specifically fatty acid β- 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

(3) evaluating hepatic fatty acid metabolic ability of the subject based on the change in the radioactive emission over time.

The above method can also be carried out by the following steps of:

(1') detecting, over time, radiation generated from a radioisotope-labeled fatty acid accumulated in the liver of a subject to whom the radioisotope-labeled fatty acid has been administered and who has 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' ) evaluating the hepatic fatty acid metabolic ability of the subject based on the change in the radioactive emission over time.

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.

As fatty acids labeled with radioisotope (radioisotope- labeled fatty acids) used herein, fatty acids in which iodine is replaced with radioactive iodine (e.g., ¹²³I) and which is structured in such a manner as to stay in liver cells for as long a time as possible after being incorporated thereinto can be mentioned. Specific examples of such 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. Among the above, ¹²³I- BMIPP is preferable.

Although there is no limitation to the routes for administering a radioisotope-labeled fatty acid to the subject, intravenous administration is preferable.

After a radioisotope-labeled fatty acid is administered to a subject, 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. For example, a gamma camera, and preferably, a gamma camera equipped with a low- energy parallel collimator can be mentioned. Subsequently, a 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.

Accordingly, by comparing time-radioactivity curves obtained from the scintigrams measured with respect to the subject before and after therapeutic _* treatment or the analystical 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. As 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;

(b) detecting, over time, radioactivity generated from the radioisotope-labeled fatty acid accumulated in the liver of the subject to whom the radioisotope-labeled fatty acid has been administered;

(c) creating a time-radioactivity curve based on the detection result, and obtaining, by a least squares method, a maximum likelihood line (abscissa: elapsed time after administration, ordinate: radiation) based on detection results ranging from 300 seconds to 1800 seconds, and preferably 500 seconds to 1800 seconds after the administration of a radioisotope-labeled fatty acid; and

(d) normalizing the time-radioactivity curve to a time-fatty acid retention curve, and calculating the gradient (%/min) of the maximum likelihood line based on the fitted curve, when estimated radioactivity at the administration of the radioisotope-labeled fatty acid determined by extraporation of the maximum likelihood line is set as fatty acid retention of 100%. The positive gradient value of the maximum likelihood line thus obtained is the hepatic clearance rate (%/min) .

As shown in Examples described later, if the fatty acid clearance rate (¹²³I-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.

Moreover, a hepatic fatty acid metabolic ability

(hepatic fatty acid β-oxidation ability) of a subject can be evaluated based on the hepatic fatty acid clearance rate (%/min) . As above, if 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.

EFFECT OF THE INVENTION 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. In particular, according to the invention, 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 β-oxidation ability disorder) . Since treatments for NASH, as a matter of course, varies according to the cause of the onset of the disease, the clarification of the cause of NASH patients or patients with a potential risk of NASH makes it possible to take appropriate measures for alleviating or retarding the progression of the NASH. The invention further provides a reagent for use in the above-described detection method, which can facilitate carrying out the method. In addition, 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.

EXAMPLES

Examples are given below to illustrate the invention in more detail, but it is to be understood that the invention is not limited thereby. [ Example 1]

Patients (15 females, 22 males, a total of 37 persons) diagnosed as having non-alcoholic steatohepatitis (NASH) according to Brunt's method (Brunt EM., Am. J Gastroenterol 1999; 94:2467) were measured for their hepatic fatty acid beta- oxidation ability using ¹²³I-BMIPP [¹²³I-labeled-15- (4-iodophenyl) - 3 (R, S) -methylpentadecanoic acid] and also checked for their genotype at the 3513rd position of the FABPl gene. These subject patients (a total of 37 persons) were such that their alcohol intake was not more than 20 g per day and 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. The patients were not being treated with methotrexate, corticoid, or insulin.

(1) ¹²³I-BMIPP scintigram and image analysis

¹²³I-BMIPP [¹²³I-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 ¹²³I-BMIPP clearance ability by ¹²³I-BMIPP scintigraphy. More specifically, the patients fasted overnight and, received an intravenously administered 20 mL of physiological saline containing 111 Mbq ¹²³I-BMIPP, and lay in a bed equipped with a gamma (scintillation) camera. The ¹²³I-BMIPP administered site was subjected to dynamic planner scanning (1 second x 60 frames, 30 lines x 60 frames) to determine the uptake of ¹²³I- 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%) .

Using the entire left ventricle, scarcely influenced by ¹²³I-BMIPP accumulation in the right kidney, as a blood pool indicator and S8 (the anterior superior subsegment) of the right hepatic lobe, scarcely influenced by ¹²³I-BMIPP accumulation in the heart, as a hepatic pool indicator, ROIs (regions of interest) were imaged on a flat screen. Fig. 1 shows the results (images) obtained by measuring the amount of gamma rays (per 1 cm² for 30 seconds) in ROIs ((1) the heart, (2) the liver) with the lapse of time. In Fig. 1, 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 ¹²³I- BMIPP moves to the liver within 1 minute after administration and the amount of ¹²³I-BMIPP in the liver peaks in about 5 minutes and then gradually declines.

Time-radioactivity curves (abscissa: time after administration of ¹²³I-BMIPP, ordinate: radioactivity) were made by plotting the amount of gamma rays (per 1 cm² 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 ¹²³I-BMIPP moves to the liver within 60 seconds after administration and the amount of ¹²³I-BMIPP in the liver and peaks within 500 seconds after administration and decreases linearly over the following 1400 seconds. Therefore, 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 ¹²³I- BMIPP were used to make a maximum likelihood line according to the least sguares method. The amount of ¹²³I-BMIPP at the time of ¹²³I-BMIPP administration was extrapolated, and this was set as 100% ¹²³I-BMIPP retention. "¹²³I-BMIPP clearance rate (%/min.)" indicating clearance of ¹²³I-BMIPP from the liver was calculated relative to the above extrapolated ¹²³I-BMIPP amount at the time of ¹²³I-BMIPP administration being set as 100% ¹²³I-BMIPP retention.

Fig. 2 shows the results showing hepatic ¹²³I-BMIPP retention (%) from immediately after to 1830 seconds after administration of ¹²³I-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 (¹²³I-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 (¹²³I-BMIPP clearance rate: less than 0.5 % min., indicated by black circles (•) in Fig. 2, 10 of the 37 patients) .

(2) Examination of the genotype at the 3513rd position of FABPl gene

Blood samples were collected from peripheral veins of the 37 NASH patients (15 females and 22 males) and placed in blood-collection tubes containing an anticoagulant. Using a commercially available DNA purification kit, 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. Subsequently, 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.

The results clearly show that among the NASH patients (37 persons) , all the NASH patients who showed an abnormally low ¹²³I-BMIPP clearance rate, i.e., less than 0.5 %/min. (10 persons), had the G/G genotype at the 3513rd position of the FABPl gene and had a polymorphism that causes a 94th amino acid change (T->A) in FABPl, in the form of a homozygote (G/G) (T94A polymorphism: AA) . In contrast, NASH patients who had a ¹²³I-BMIPP clearance rate of no less than 0.5 %/min. (27 persons) had either the A/G (G/A) (T94A polymorphism : AT) or A/A (>T94A polymorphism : TT) genotype at the 3513rd position of the FABPl gene (Fig. 3) . (3) Medical treatment and evaluation of effects

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. On the 246^th day of administration, ¹²³I-BMIPP scintigraphy was performed to make time-radioactivity curves and time-BMIPP retention curves in the same manner as in (1) above and ¹²³I-BMIPP clearance rates (%/min) were determined. Fig. 4 shows one example of the results. In Fig. 4, the graph indicated by black dots (♦) is a time-BMIPP retention curve obtained by measurement before the administration of the fibrate drug, whereas the graph indicated by white dots (O) is a time-BMIPP retention curve obtained by measurement after the administration of the fibrate drug. As shown in Fig. 4, the ¹²³I-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.

[Example 2]

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.

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 ¹²³I- BMIPP [¹²³I-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 (a total of 10 persons) 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. 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

Table 1, all cases (10 persons) showed an abnormally low 123 I- BMIPP clearance rate, i.e., less than 0.5 %/min.

[Example 3]

The 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.

<Tests>

(1) 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/).

(2) Computed tomography (measurement of visceral fat occupying area and subcutaneous fat occupying area)

Computed tomography (CT) 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.

(3) Determination of body mass index (BMI) Weight and height were measured for all the patients to determine their body mass index (BMI) [weight (kg) /(height (m))²]. Patients with a BMI of at least 30 and those with a BMI of less than 30 were classified as obese and non-obese, respectively according to the standard WHO definition.

(4) Therapeutic effects of the administration of a fibrate drug

As mentioned above, tamoxifen, a potent antagonist of estrogen, often causes non-alcoholic steatohepatitis (NASH) in breast cancer patients, but it is kn©wn that fatty liver of such patients (tamoxifen-induced NASH patients) is successfully ameliorated by fibrate drugs (e.g., Bezafibrate) known to activate PPAR-α (peroxisome proliferator-activated receptor-α) (Saibara T, Onishi S, Ogawa Y, Yoshida S, Enzan H, Bezafibrate for tamoxifen-induced non-alcoholic steatohepatitis, Lancet. 999; 353 (9166): 1802).

The results of Example 2 show that all the tamoxifen- induced NASH patients had reduced hepatic fatty acid beta- oxidation abilities (¹²³I-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 ¹²³I-BMIPP clearance rate determined from CT (computed tomography) and ¹²³I-BMIPP scintigraphy, respectively, after 1 year of administration were compared to those determined before the administration to evaluate therapeutic effects of the fibrate drug.

(a) Relationship between the body mass index and ¹²³I-BMIPP clearance rate

The non-alcoholic steatohepatitis (NASH) patients (37 persons) were classified into the following 4 groups, based on the ¹²³I-BMIPP clearance rates determined in Example 1 and the body mass index (BMI) determined in (3) above (Fig. 5) . A ¹²³I- BMIPP clearance rate of at least 0.5%/min. is defined as a "high ¹²³I-BMIPP clearance rate", and a ¹²³I-BMIPP clearance rate of less than 0.5%/min. as a "low ¹²³I-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 ¹²³I-BMIPP clearance rate (12 persons) Group B: Non-obese NASH patients with a high ¹²³I-BMIPP clearance rate (15 persons) > Group C: Non-obese NASH patients with a low ¹²³I-BMIPP clearance rate (9 persons)

Group D: Obese NASH patients with a low ¹²³I-BMIPP clearance rate (1 person)

As classified above, among the 24 non-obese NASH patients (groups B and C) , 9 persons had a low ¹²³I-BMIPP clearance rate (group C) ; among the 13 non-obese NASH patients

(groups A and D) ,

1 person had a low ¹²³I-BMIPP clearance rate (group D) .

₍b₎ Clinical characteristics and ¹²³I-BMIPP clearance rate

Table 2 shows the summary of clinical characteristics of the above groups A to C. [Table 2]

(*: p<0.05, **: p<0.02, #■ p<0.0l) As shown in Table 2, compared to group B consisting of non-obese NASH patients, 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 ¹²³I- BMIPP clearance rate) . However, 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 ¹²³I-BMIPP clearance rates) had a visceral fat occupying area of 138.7 ± 51.8 cm², 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 cm², i.e., all high values, but group C had a BMI of 25.4 ± 2.9 kg/m², i.e., a low value, unlike group A consisting of obese NASH patients.

There are few differences in clinical findings between groups B and C, both consisting of non-obese NASH patients, (although group B had a high fatty acid clearance rate, whereas group C had a low fatty acid clearance rate) . However, these two groups were clearly different in terms of mean liver/spleen ratio and fat occupying area (p< 0.02).

(c) Relationship between liver specimen pathological features and BMI (body mass index) Fig. 6 shows the relationships between the body mass index (BMI) (kg/m²) and fat occupying area (%) obtained from liver biopsy specimens from the 37 NASH patients.

The symbol Φ indicates the results of group C patients (non-obese NASH patients with a low ¹²³I-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.

As shown in (a) to (c),»except for one case (group D) , all the obese NASH patients had a ¹²³I-BMIPP clearance rate of at least 0.5 %/min (group A). In contrast, among the 24 non-obese NASH patients, 9 persons (38%) had a ¹²³I-BMIPP clearance rate of less than 0.5 %/min (group C). A large amount of fat accumulation in the liver was observed in these patients. It can be explained therefrom that since excess free fatty acid competitively inhibits ¹²³I-BMIPP clearance in cases showing a low ¹²³I-BMIPP clearance rate (groups C and D) , fat accumulation in liver cells progresses.

Among the 9 persons of group C, 5 persons had a body mass index of 25 or less, i.e., a low value. However, since liver cell mitochondria are often damaged by NASH, it would be no surprise if these 5 persons suffered from mitochondria disorders in the future. Among the 9 persons of group C, 2 persons were patients who had developed NASH due to the administration of tamoxifen (tamoxifen-induced NASH) , and the remaining seven all had a variant FABPl gene (a3513g) in the form of a homozygote (G/G) . Of the obese NASH patients (13 persons, groups A + D) , two cases had the variant FABPl gene (a3513g) in the form of a homozygote (G/G) , and one of them (group D) showed a ¹²³I-BMIPP clearance rate of less than 0.5 %/min.

Although there was no clinically significant difference between the non-obese NASH patients of group C (9 persons) with a ¹²³I-BMIPP clearance rate of less than 0.5 %/min. or and the non- obese NASH patients of group B (15 persons) with a ¹²³I-BMIPP clearance rate of at least 0.5 %/min., more serious fatty liver was observed in the former group than in the latter (p< 0.02) (Table 2) . This result shows that evaluation of ¹²³I-BMIPP clearance is a highly effective method for identifying non-obese NASH patients who have a background serious reduction in hepatic fatty acid metabolism. Since obese NASH patients with seriously reduced hepatic fatty acid beta-oxidation abilities cannot sufficiently oxidize excess fatty acid which results in accumulation, NASH progresses rapidly. Therefore, beta-oxidation promoters (e.g., fibrate drugs) are considered to be highly effective in such patients. (d) Therapeutic effect of fibrate drug in tamoxifen- induced NASH patients

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.

As shown in 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. Before the treatment with Benzafibrate, the patients (3 persons) had ¹²³I-BMIPP clearance rates of 0.237 %/min., 0.304 %/min., and 0.306 %/min., respectively. After the treatment with Benzafibrate, their ¹²³I-BMIPP clearance rates were remarkably increased (0.625%/min. , 0.917%/min., and

0.818%/min.) without change in their BMI. This shows that the administration of Benzafibrate increases hepatic fatty acid beta- oxidation ability. 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 ¹²³I-BMIPP clearance rate using ¹²³I-BMIPP scintigraphy.

Consideration of Examples 1 to 3>

Recently, obesity has increased worldwide and metabolic syndromes, including non-alcoholic fatty liver, and non-alcoholic steatohepatitis, have become a worldwide social issue. Since serious fatty liver is an essential εlinical characteristic of NASH, research into hepatic fatty acid metabolism is urgently required. ¹²³I-BMIPP, which has the characteristic of being metabolized with difficulty by myocardial mitochondria, has been conventionally used as the most suitable imaging agent for imaging damaged areas in cardiac muscle. However, since ¹²³I-BMIPP is easily metabolized in hepatic mitochondria, there has been no attempt to use ¹²³I-BMIPP scintigraphy to evaluate the functions of mitochondria in acute and chronic liver diseases.

As shown in Examples 1 and 2, hepatic ¹²³I-BMIPP clearance was investigated using ¹²³I-BMIPP scintigraphy and the obtained time-radioactivity curves confirmed that ¹²³I-BMIPP was uptaken by the liver within 1 minute after administration of ¹²³I- BMIPP. The ¹²³I-BMIPP concentration in the liver peaked within 500 seconds after administration of ¹²³I-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 ¹²³I-BMIPP clearance rates can be analyzed in a reproducible manner using ¹²³I-BMIPP scintigraphy. Since the evaluation of internal fatty acid beta-oxidation ability using ¹²³I-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. In contrast, 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.

The results show that the frequency of appearance of homozygous guanine (G/G) at the 3513rd position of the FABPl gene in the NASH patients was such that the odds ratio was 4.81 (95% confidence interval: 2.40-9.62) and the level of significance was p = 2.53 x 10^"6, showing that its frequency of appearance is significantly high in NASH patients. The frequency of possession of the variant FABPl gene (a3513g) in the NASH patients was such that the odds ratio was 2.18 (95% confidence interval: 1.51-3.15) and the level of significance was p = 2.62 x 10^~5, showing that the frequency of possession of the FABPl variant (a3513g) is significantly higher in NASH patients than in healthy persons. Therefore, homozygous guanine (G/G) at the 3513rd position of the FABPl gene is suggested as a risk factor for non-alcoholic steatohepatitis (NASH) .

[Example 5]

Fifty-eight persons with a history of myocardial infarction (myocardial infarction cases) 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.

The results show that the frequency of appearance of homozygous guanine (G/G) at the 3513rd position (SNP3513) of the FABPl gene in the myocardial infarction cases was such that the odds ratio was 5.51 (95% confidence interval: 2.42-12.53) and the level of significance was p = 1.10 x 10^~5, showing that the frequency of appearance is significantly high in myocardial infarction cases. The frequency of possession of the variant FABPl gene (a3513g) in the myocardial infarction cases was such that the odds ratio was 3.03 (95% confidence interval: 1.89-4.85) and the level of significance was p = 2.05 x 10^"6, showing that the frequency of possession of the FABPl variant (a3513g) is significantly higher in myocardial infarction cases than in healthy persons. Therefore, homozygous guanine (G/G) at the 3513rd position (SNP3513) of the FABPl gene is suggested as a risk factor for myocardial infarction.

Claims

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) ] .

2. The method according to claim 1, wherein the genetic polymorphism is a homozygotic genetic polymorphism in the FABPl gene.

3. The method according to claim 1, wherein the lipid metabolism disorder or disease caused thereby is a hepatic fatty acid beta-oxidation ability disorder or disease caused thereby.

4. The method according to claim 3, wherein the disease caused by the hepatic fatty acid beta-oxidation ability disorder is non-alcoholic steatohepatitis, fatty liver or myocardial infarction.

5. A method for detecting a lipid metabolism disorder or a risk of disease caused thereby, comprising the following steps (a) to (c) :

(a) extracting genomic DNA from a biological sample of a subject; (b) detecting the 3513rd base of the base sequence of the FABPl gene contained in the extracted genomic DNA; and (c) identifying whether the base is A or G.

6. The method according to claim 5, further comprising the following step (d) : (d) if the base is homozygous for G (G/G) , determining that the subject has a lipid metabolism disorder or a risk of a disease caused thereby.

7. 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; (ii) calculating a fatty acid clearance rate (%/min) in the liver from the change in the radioactivity detected over time in step (i) ; and

8. The method according to claim 7, 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.

9. 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.

10. A primer comprising the labeled or unlabeled oligonucleotide according to claim 9, the primer being used for determining a lipid metabolism disorder or a risk of a disease caused thereby.

11. 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 .

12. A probe comprising the labeled or unlabeled oligo- or polynucleotide according to claim 11, the probe being used for determining a lipid metabolism disorder or a risk of a disease caused thereby.

13. A reagent for detecting a lipid metabolism disorder or a risk of a disease caused thereby, the reagent comprising the primer according to claim 10 and/or the probe according to claim 12; or a kit comprising at least one of the reagents .

14. 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 FABPl.

15. The method according to claim 14, wherein the genetic polymorphism is a homozygotic genetic polymorphism in the FABPl gene.

16. The method according to claim 14, which is a method for detecting the presence or absence of the genetic polymorphism using change (T->A) of the 94th amino acid of FABPl as an index .

17. 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) :

18. The method according to claim 17, further comprising the following step (d) : ► (d) if the base is homozygous for G (G/G) , determining that the NASH is of hepatic fatty acid beta-oxidation ability disorder-type .

19. 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) :

(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; (ii) calculating a fatty acid clearance rate (%/min) in the liver, from the change in the radioactivity detected over time in step (i) ; and

(iii) determining whether the fatty acid clearance rate is less than 0.5%/min or not less than 0.5%/min.

20. The method according to claim 19, further comprising the following step (iv) :

(iv) if the fatty acid clearance rate is less than

0.5%/min, determining that the NASH is of hepatic fatty acid beta-oxidation ability disorder-type; and if the fatty acid clearance rate is not less than 0.5%/min, determining that the

NASH is of normal hepatic fatty acid beta-oxidation-type.

21. A primer comprising the following labeled or 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 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. ►

22. 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 nucleotide of the base sequence.

23. A reagent for detecting NASH of hepatic fatty acid beta-oxidation ability disorder-type, the reagent comprising the primer according to claim 21 and/or the probe according to claim 22; or a kit comprising the reagent.

24. 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) .

25. A method of reducing the risk of developing NASH, and in particular NASH of hepatic fatty acid beta-oxidation ability disorder-type, in a subject having in the FABPl gene SNP3513 that is homozygous for G (G/G) , comprising administering a fibrate drug to the subject.

26. 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.

27. A method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease, the method 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;

28. A method for screening for a component effective for alleviating lipid metabolism disorder or reducing risk of developing lipid metabolism disorder-related disease, the method comprising the following steps of:

29. 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, wherein hepatic fatty acid metabolic abilities are compared before and after the treatment. t

30. The method according to claim 29, wherein the hepatic fatty acid metabolic ability is measured by a method comprising the following steps (a) and (b) :

(a) administering a radioisotope-labeled fatty acid to a subject, and detecting, over time, radiation generated from the radioisotope-labeled fatty acid accumulated in the liver; and

(b) determining hepatic fatty acid metabolic ability based on the change in the radiation detected over time in step

(a).

31. 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, the method comprising the following steps of: