JP2010022293A - Method for examination of non-alcoholic fatty liver disorder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an examination method for estimating non-alcoholic fatty liver disorder. <P>SOLUTION: The method includes analyzing a single nucleotide polymorphism of a base corresponding to the base No.61 of a specific base sequence on an iNOS gene or a single nucleotide polymorphism of a base of the state of linkage disequilibrium with the base and examining non-alcoholic fatty liver disorder based on the analytic results. Also, a probe for the examination of non-alcoholic fatty liver disorder having a sequence of ≥10 bases including the base No.61 in a specific base sequence or its complimentary sequence, and a primer for the examination of non-alcoholic fatty liver disorder capable of amplifying a region including the base No.61 in a specific base sequence are provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a test method for non-alcoholic fatty liver disease and a probe or primer used therefor.

Nonalcoholic fatty liver disorder (NAFLD) is now recognized as one of the important health problems. NAFLD has a wide range of symptoms ranging from simple fatty liver to nonalcoholic steatohepatitis (NASH). NAFLD has no subjective symptoms and is difficult to determine. Although NAFLD is speculated on the basis of various clinical aspects (increased ALT, obesity and presence of type 2 diabetes), these clinical aspects are not sufficient to establish the diagnosis and prediction of NAFLD. At present, the boundary between NAFLD and NASH can also be determined only by biopsy of the liver and cannot be predicted by other testing methods. Excessive fat accumulation in the liver is observed in 20-30% of the population of developed countries, and about 1-3% of the population develops from excessive fat accumulation in the liver, ie from fatty liver to NASH. .
Genetic factors are considered to be important for the progression of NAFLD as well as environmental factors (Non-Patent Documents 1 to 4). However, there are only a few genetic research reports.

Inducible nitric oxide synthase (iNOS) is induced by inflammation and stress, synthesizes free radical nitric oxide, and is involved in various pathological conditions. There are reports that iNOS is highly expressed in severe NASH patients (Non-patent Document 5) and that iNOS knockout mice are susceptible to liver fibrosis due to a high-fat diet (Non-patent Document 6). There is also a report that a selective inhibitor of iNOS suppressed fibrosis in a rat cirrhosis model (Non-patent Document 7).
However, there is no report that a single nucleotide polymorphism of the iNOS gene is associated with NAFLD.
Namikawa C, Shu-Ping Z, Vyselaar JR, Nozaki Y, Nemoto Y, et al. Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis. J Hepatol. 2004; 40: 781-6 Gambino R, Cassader M, Pagano G, Durazzo M, Musso G. Polymorphism in microsomal triglyceride transfer protein: a link between liver disease and atherogenic postprandial lipid profile in NASH? Hepatology. 2007; 45: 1097-107. Tokushige K, Takakura M, Tsuchiya-Matsushita N, Taniai M, Hashimoto E, et al. Influence of TNF gene polymorphisms in Japanese patients with NASH and simple steatosis. J Hepatol. 2007; 46: 1104-10. Merriman RB, Aouizerat BE, Bass NM. Genetic influences in nonalcoholic fatty liver disease.J Clin Gastroenterol. 2006; 40: S30-3. Garcia-Monzon C, Martin-Perez E, Iacono OL, Fernandez-Bermejo M, Majano PL, Apolinario A, Larranaga E, Moreno-Otero R. Characterization of pathogenic and prognostic factors of nonalcoholic steatohepetitis associated with obesity. J Hepatol 2000; 33 : 716-724 Chen Y, Hozawa S, Sawamura S Sato S, Fukuyama N, Tsuji C, Mine T, Okada Y, Tanino R, Osugi Y, Nakazawa H. Defiency of inducible nitric oxide synthase exacerbates hepatic fibrosis in mice fed high-fat diet.Biochem Biophys Res Commun 2005; 326: 45-51 Kikuchi H, Katsuramaki T, Kukita K, Taketani S, Meguro M, Nagayama M, Isobe M, Mizuguchu T, Hirata K. New straregy for the antifibrotic therapy woth oral administration of FR260330 (a slective inducible nitiric oxide synthase inhibitor) in rat experimental liver cirrhosis. Wound Repair Regen 2007; 15: 881-888

  An object of the present invention is to provide a test method for accurately predicting the onset and progression of NAFLD.

  The present inventors have made extensive studies to solve the above problems. As a result, it was found that a single nucleotide polymorphism of a specific base present on the iNOS gene or a single nucleotide polymorphism of a base in linkage disequilibrium with the base is related to NAFLD, and the present invention has been completed.

That is, the present invention is as follows.
(1) A single nucleotide polymorphism in the base corresponding to the 61st base of the nucleotide sequence of any one of SEQ ID NOs: 1 to 4 on the iNOS gene or a single nucleotide polymorphism of a base in linkage disequilibrium with the base And testing non-alcoholic fatty liver disorder based on the analysis result.
(2) A non-alcoholic fatty liver injury test probe having a sequence of 10 bases or more including the base at the 61st base in the base sequence of any one of SEQ ID NOs: 1 to 4 or a complementary sequence thereof.
(3) A non-alcoholic fatty liver injury test primer capable of amplifying a region containing the 61st base in the base sequence of any one of SEQ ID NOs: 1 to 4.

Since the onset risk and progression of NAFLD can be accurately and non-invasively predicted by the test method of the present invention, NAFLD can be prevented or progress can be suppressed. In addition, iNOS polymorphisms can be an excellent index for determining the risk of side effects such as the onset or exacerbation of NAFLD or NASH when an iNOS inhibitor is administered to a person who has a predisposition to becoming NAFLD (SNP). .

<1> Test Method of the Present Invention The test method of the present invention comprises a single nucleotide polymorphism (SNP) present on an inducible nitric oxide synthase (iNOS) gene or a base in linkage disequilibrium with the base. This is a method for analyzing a single nucleotide polymorphism of NAF and examining NAFLD based on the analysis. NAFLD means infectious hepatitis, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, hemochromatosis, α ₁ -antitrypsin deficiency, Wilson disease, drug hepatitis or alcoholic hepatitis, and It refers to a pathological condition diagnosed according to the criteria of, for example, Sanyal AJ. AGA technical review on nonalcoholic fatty liver disease. Gastroenterol. 2002: 123: 1705-25. In the present invention, “inspection” includes an inspection for predicting whether or not NAFLD will occur in the future, and an inspection for predicting whether or not the degree of NAFLD will deteriorate (for example, progress to NASH). .

The iNOS gene is preferably a human iNOS gene, and examples thereof include a gene having a sequence registered in GenBank Accession No. NM_000625. However, the gene is not limited to the gene of the above sequence because substitution or deletion may exist in one or a plurality of bases due to differences in race.

  SNP-1 to SNP-4 shown below are listed as single nucleotide polymorphisms of the iNOS gene related to NAFLD.

In Table 1, for example, SNP-1 means a polymorphism of cytosine (C) / thymine (T) in the 827748th base of GenBank Accession No. NT_010799.14, and when this base is T, N
There is a high probability of becoming AFLD. Moreover, when analyzing in consideration of alleles, there is a high probability that SNP-1 becomes NAFLD in the order of TT>TC> CC. SNP-1 is an SNP present in intron 21 of the iNOS gene. dbSNP indicates the registration number of the dbSNP database (//www.ncbi.nlm.nih.gov/projects/SNP/) of National Center for Biotechnology Information.
The same applies to other SNPs, but the right base is a base that is likely to be NAFLD in all cases (for example, in the case of SNP-2, the case of C in T / C is likely to be NAFLD, and SNP-3 In the case of A / G, G is likely to be NAFLD, and in the case of SNP-4, T of C / T is likely to be NAFLD).
For SNP-1 to SNP-4, sequences having a total length of 121 bp including the SNP base and the region of 60 bp before and after that are shown in SEQ ID NOs: 1 to 4, respectively. Each 61st base has a polymorphism.
Bases corresponding to these bases are analyzed in the present invention. Here, “corresponding” means a corresponding base in the region having the sequence on the human iNOS gene, and even if the sequence is slightly changed at a position other than the SNP due to a difference in race, It also includes analyzing the corresponding base in it.

NAFLD can be examined by examining the type of base of the SNP. The number of SNPs to be inspected may be one type or plural (haplotype analysis). If SNP-1 is C, the other SNPs are the left bases (SNP-2 = T, SNP-3 = A, SNP-4 = C), and the SNP types correlate with each other. Therefore, sufficiently accurate NAFLD prediction can be performed by analyzing only one location. As for the iNOS gene sequence, the sense strand may be analyzed, or the antisense strand may be analyzed.
In addition, the base to be analyzed in the present invention is not limited to the above, and a polymorphism of a base in linkage disequilibrium with the above base may be analyzed. Here, the “base in linkage disequilibrium with the above-mentioned base” refers to a base that satisfies the relationship of r ² > 0.5 with the above-mentioned base.

  The sample used for the analysis of the single nucleotide polymorphism of the iNOS gene is not particularly limited as long as it is a sample containing chromosomal DNA. Examples thereof include body fluid samples such as blood and urine, body hair such as cells and hair, and nails. . Although these samples can be used directly for analysis of single nucleotide polymorphisms, it is preferred to isolate chromosomal DNA from these samples by a conventional method and analyze them.

  Analysis of a single nucleotide polymorphism of the iNOS gene can be performed by a usual single nucleotide polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, and the like.

The sequence can be performed by a usual method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of a base showing polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. When sequencing is performed, it is preferable to amplify a fragment containing a polymorphism in advance by PCR or the like.

  It can also be analyzed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a polymorphism and corresponding to each polymorphism is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of an amplification product.

It is also possible to amplify a DNA fragment containing a polymorphism and determine which type of polymorphism is based on the difference in mobility in electrophoresis of the amplified product. Examples of such a method include a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing a polymorphic site of the iNOS gene is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

  Furthermore, when a base showing polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, first, a DNA sample is amplified by PCR and cut with a restriction enzyme. The DNA fragments can then be separated and the type of polymorphism can be determined by the size of the detected DNA fragment.

  It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, it is possible to determine which base an SNP is by preparing probes corresponding to each base and examining which probe hybridizes.

<2> Reagent for test | inspection of this invention This invention also provides test reagents, such as a primer and a probe for test | inspecting NAFLD. Examples of such a probe include a probe that includes the polymorphic site in the iNOS gene and can determine the type of base at the polymorphic site based on the presence or absence of hybridization. Specifically, a probe having a length of 15 bases or more having a sequence containing the 61st base of any one of SEQ ID NOs: 1 to 4 or a complementary sequence thereof can be mentioned. The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases. Examples of such probes include probes of SEQ ID NO: 11 and SEQ ID NO: 12 (SNP-3), probes of SEQ ID NO: 13 and SEQ ID NO: 14 (SNP-4), and the like. SNP-1 and SNP-2 can also be designed based on SEQ ID NOs: 1 and 2.

Examples of the primer include a primer that can be used for PCR for amplifying the polymorphic site in the iNOS gene, or a primer that can be used for sequence analysis (sequencing) of the polymorphic site. Specifically, a primer that can amplify or sequence the region containing the 61st base of any one of the nucleotide sequences of SEQ ID NOS: 1 to 4 can be mentioned. The length of such a primer is preferably 15 to 50 bases, more preferably 20 to 35 bases. Such primers can be set, for example, at positions upstream or downstream of the polymorphic site in SEQ ID NOs: 1-4.
As a primer for sequencing the polymorphic site, a primer having a sequence 5′-side of the polymorphic base, preferably 30 to 100 bases upstream, or 3 ′ of the polymorphic base is used.
Examples include a primer having a sequence complementary to a side region, preferably a region downstream of 30 to 100 bases. Primers used to determine polymorphism based on the presence or absence of amplification by PCR have a sequence containing the polymorphic base, a primer containing the polymorphic base on the 3 ′ side, or a sequence containing the polymorphic base. Examples thereof include a primer having a complementary sequence and containing a complementary base of the polymorphic base on the 3 ′ side.
The test reagent of the present invention may contain PCR polymerases, buffers, hybridization reagents, etc. in addition to these primers and probes.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Materials and Methods Subjects 115 Japanese NAFLD patients (65 NASH and 50 simple fatty liver) and 435 healthy control subjects were recruited at Yokohama City University Hospital.
All control subjects were confirmed to have normal liver function, no viral hepatitis infection, and no alcoholism. Control subjects had BMI <25 kg / m ² , fasting blood glucose (<110 mg / dl), neutral fat (<150 mg / dl), HDL cholesterol (> 40 mg / dl) normal, and systolic blood pressure (<130 mmHg) and diastolic blood pressure (<85 mmHg) were also normal.
On the other hand, all 115 patients were confirmed as NAFLD by liver biopsy. That is, liver biopsy tissue was stained with hematoxylin-eosin, reticuline, and Masson trichrome stain and examined by two pathologists. The histological criteria for the diagnosis of NAFLD is the presence of large lipid steatosis of hepatocytes, with nuclear movement to the cell edge (Sanyal AJ.
AGA technical review on nonalcoholic fatty liver disease. Gastroenterol. 2002: 123: 1705-25). Based on this standard, NAFLD was diagnosed. If greater than 5% of hepatocytes were found to have large lipid steatosis, the patient was classified as either simple fatty liver or NASH. The criteria for diagnosis of NASH include hepatocyte macrophageous lipidation, intralobular inflammation, balloon-like swelling of hepatocytes in zone 3 (also called balloon-like swelling), pericentral fibrosis, Fibrosis is present (Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, et al. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology. 1999; 116: 1413-1419 .; MR, James OF, Burt AD, Bennett MK, Day CP. The natural history of nonalcoholic fatty liver: a follow-up study. Hepatology. 1995; 22: 1714-1719.).
Patients with the following diseases were excluded from this study: infectious hepatitis (B and C, Epstein-Barr virus infection), autoimmune hepatitis, primary biliary cirrhosis (PBC), sclerosis Cholangitis, hemochromatosis, α ₁ -antitrypsin deficiency, Wilson's disease, drug-induced hepatitis, alcoholic hepatitis, and heavy drinkers (those with current or past daily alcohol consumption> 20 g).
None of the NAFLD patients as subjects had clinical findings of liver decompensation (such as hepatic encephalopathy, ascites, variceal bleeding, or serum bilirubin concentration exceeding twice the normal upper limit).
Written informed consent was obtained from all subjects prior to participation in the study. The research protocol complies with the ethical guidelines of the Declaration of Helsinki in 1975, and has been approved by the Yokohama City Hospital and the RIKEN Research Committee.

Clinical and laboratory evaluations Venous blood samples are taken from subjects after an overnight fast (12 hours), and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, Hemoglobin A1c (HbA1c), immunoreactive insulin, total cholesterol, and neutral fat were measured. The biochemical parameters of all experiments were measured with an automated analyzer.

DNA preparation and SNP genotyping Genomic DNA was obtained from each blood sample with Genomix (Talent SRL, Trieste, Italy).
). SNPs in iNOS were selected from the SNP database of IMS-JST (Institute for Medical Science-Japan Science and Technology Agency). Ten SNPs were selected in which the minor allele frequency exceeded 0.1 and the observed genetic polymorphism (eg, TT, CT, CC) frequency did not deviate significantly from the Hardy-Weinberg equilibrium ( P> 0.001). Invader probes (Third Wave Technologies, Madison, Wis.) Were synthesized for these SNPs, and SNPs in patients and controls were genotyped by a combination of previously reported multiplex PCR and invader assays (Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y. A high-throughput SNP typing system for genome-wide association studies. J Hum Genet 2001; 46: 471-477).

Statistical analysis For each NAFLD patient-control comparison, a chi ² test was used to compare genotype or allele frequencies between NAFLD patients and controls in three different modes. In the first mode (allele frequency mode), allele frequencies were compared between NAFLD patients and controls using a 2 × 2 contingency table. In the second mode (recessive mode), the frequency of subjects who are homozygous for allele 1 is compared with other subjects using a 2 × 2 contingency table, and the third mode (dominant mode) Then, using a 2 × 2 contingency table, the frequency of subjects having allele 1 (homozygosity and heterozygosity of allele 1) was compared with other subjects. The odds ratio and its 95% confidence interval (CI) were calculated by the Wolff's method. Hardy-Weinberg equilibrium was evaluated using the χ ² test. Haplotype blocks were determined using Haploview 3.2 (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005; 21: 263-265. ). Clinical data were expressed as mean ± standard deviation (SD). Clinical parameters between NAFLD patient group and control group were compared by Student t test.

Results NAFLD patient-control correlation analysis Table 2 shows the clinical data of NAFLD patient group and control group.

From the IMS-JST SNP database, select 10 SNPs (rs3794756, rs2255929, rs2297510, rs2297511, rs2297512, rs1060822, rs2297516, rs2248814, rs2297520, rs3794764) within the iNOS gene with a minor allele frequency greater than 0.1; The correlation with NAFLD was examined. As a result, among the 10 SNPs, 4 SNPs (rs2297510, rs2297511, rs2297512, rs1060822) were significantly correlated, and these showed the lowest P value in the recessive model (P = 0.0008, table). 3). The OR (95% confidence interval) is 0.70 (0.51-0.97) in the recessive model, thus indicating that the major allele is protective against the development of NAFLD.
For rs3794756, rs2255929, rs2297516, rs2248814, rs2297520, and rs3794764, the sequences including SNPs and 60 bp before and after thereof are shown in SEQ ID NOs: 5 to 10, respectively.

Furthermore, rs2297510 and rs1060822 were examined in detail (Table 4).
Divide the NAFLD group into NASH and simple stearosis, rs2297510
And rs1060822 were used to perform NAFLD patient-control correlation analysis. The frequency of allele 1 homozygotes at rs2297510 and rs1060822 was significantly lower in NASH subjects compared to control subjects. When comparing the frequency of allele 1 homozygotes in NASH patients with simple fatty liver subjects, the frequency between these groups was not significant. Therefore, rs2297510 and rs1060822 were significantly correlated with NAFLD combined with NASH and simple fatty liver.

Further, linkage disequilibrium analysis revealed that the above four SNPs (rs2297510, rs2297511, rs2297512, rs1060822) were in absolute linkage disequilibrium (r ² = 1) (Table 5).

Discussion The NAFLD phenotype varies from asymptomatic to liver cirrhosis with liver complications and hepatocellular carcinoma. Currently, liver biopsy is the only method that can evaluate the diagnosis and prediction of NAFLD. This time, we found that polymorphisms in the iNOS gene are related to NAFLD. These polymorphisms were considered to be useful tools for the diagnosis and / or prediction of NAFLD.

Claims (3)

Analyzes a single nucleotide polymorphism in the base corresponding to the base number 61 in the nucleotide sequence of any one of SEQ ID NOs: 1 to 4 on the iNOS gene, or a single nucleotide polymorphism of the base in linkage disequilibrium with the base. And a method for examining nonalcoholic fatty liver disorder based on the analysis result. A non-alcoholic fatty liver injury test probe having a sequence of 10 bases or more including the 61st base in the base sequence of any one of SEQ ID NOs: 1 to 4 or a complementary sequence thereof. A primer for non-alcoholic fatty liver injury test, which can amplify a region containing the base at position 61 in any one of SEQ ID NOs: 1 to 4.