KR20060096144A - SINGLE NUCLEOTIDE POLYMORPHISM OF HNF-4alpha; GENE AND USE THEREOF - Google Patents

Abstract

The present invention relates to a single-base polymorphism of the hepatocyte nuclear factor (HNF) -4α gene and its use, and more specifically, to the adenine (A) in the 179th base of the base sequence of HNF-4α of SEQ ID NO: 1. Prediction of adverse drug reactions due to individual differences in the enzyme activity of the cytochrome P450 2D6 gene (hereinafter referred to as 'CYP2D6'), or the lack of the CYP2D6 enzyme Diagnosis can be made. In addition, since HNF-4α is closely related to diabetes, the single nucleotide polymorphism of HNF-4α of the present invention may be useful for elucidating the mechanism of diabetes and / or for predicting the possibility of diabetes.

HNF-4α, Monobasic Polymorphism, Drug Metabolism Enzyme

Description

Single nucleotide polymorphism of HNF-4α gene and use approximately}

1 is a result of analyzing the single base polymorphism of HNF-4α using an automatic sequencer. The arrow indicates the position where guanine (G) is substituted with adenine (A).

Figure 2 shows some regions of the heterozygous variant gene of HNF-4α comprising a single nucleotide polymorphic site and amino acid sequences encoded therefrom. The red underline indicates the displaced position and the blue arrow indicates the start of the second exon.

Figure 3 shows the results of analyzing the CYP2D6 promoter activity by the mutant gene of HNF-4α using the luciferase reporter gene.

Lane 1: pGL2 vector

Lane 2: pGL2 vector + wild type HNF-4α

Lane 3: pGL2-CYP2D6 vector

Lane 4: pGL2-CYP2D6 vector + 0.05 μg wild type HNF-4α

Lane 5: pGL2-CYP2D6 vector + 0.1 μg wild type HNF-4α

Lane 6: pGL2-CYP2D6 vector + 0.3 g of wild-type HNF-4α

Lane 7: pGL2-CYP2D6 vector + G60D variant HNF-4α 0.05 μg

Lane 8: 0.1 μg of pGL2-CYP2D6 vector + G60D variant HNF-4α

Lane 9: 0.3 g of pGL2-CYP2D6 vector + G60D variant HNF-4α

Figure 4 shows the results of analysis of the binding degree of HNF-4α mutant protein and CYP2D6 promoter by EMSA.

Lane 1: negative control

Lane 2: HNF-4α wild type protein + CYP2D6 probe

Lane 3: HNF-4α wild type protein + CYP2D6 probe + CYP2D6 1 pmol competitor

Lane 4: HNF-4α wild type protein + CYP2D6 probe + CYP2D6 10 pmol competitor

Lane 5: HNF-4 α G60D variant protein + CYP2D6 probe

Lane 6: HNF-4α G60D variant protein + CYP2D6 probe + CYP2D6 1 pmol competitor

Lane 7: HNF-4α G60D variant protein + CYP2D6 probe + CYP2D6 10 pmol competitor

The present invention relates to a single nucleotide polymorphism of the HNF-4α gene and its use, and more particularly, a base in which the 179th base of the base sequence of HNF-4α of SEQ ID NO: 1 is substituted with adenine (A) in guanine (G). It relates to a polynucleotide containing and the use thereof.

Some of the patients on the same medication have excellent therapeutic effects, but in some patients they are less effective, or even some patients experience a serious adverse drug reaction. Individual differences in drug response can have serious medical and social health consequences from adverse drug reactions. A good example of this is that in the United States, two million Americans who normally take prescribed medications experience severe adverse drug reactions each year, of which 100,000 die from death (fourth to sixth of the causes of death in the United States). The results of a medical association. These results are due to the fact that individual drug reactions vary according to genetic and acquired factors. Acquired factors include, as is well known, differences in the characteristics of the disease itself, as well as the age, sex, diet, disorders of liver function, and drug interactions of patients directly affecting the pharmacokinetics and pharmacodynamics of the drug. However, genetic factors, compared with the acquired factors, are known to cause greater individual differences in drug response in terms of drug safety and efficacy. Therefore, researches on the differences of various drug reactions due to genetic differences of individuals are being conducted, and the concept of drug genetics is currently used in the development of new drugs. The study of clinical drug genetics for the application of customized drug therapy can be divided into three stages. The first step is to look for gene mutations that cause differences in drug responses (e.g., monobasic polymorphism (SNP), tandem repeat, etc.) Correlation studies can be used to assess the effects on drug responses. Finally, the development of a diagnostic method for high throughput screening of these clinically important genetic variants.

Coding SNPs (cSNPs), which result in amino acid mutations, are estimated to be between 50,000 and 100,000, which is the form of major genetic variations that lead to individual differences in drug response through variations in actual amino acid production. Many pharmacogenetic studies have been conducted to investigate the correlation between these monobasic polymorphisms and drug responses, to predict the individual's drug response (therapeutic effect, adverse drug reaction) and to use it as an indicator for personalized drug therapy. have. Many causes of pharmacokinetic variation among individuals are known to be due to genetic polymorphism of drug metabolism enzymes or drug transport proteins.

Many drug metabolic enzymes are genetically polymorphic, and studies of cytochrome P450 (CYP) enzymes involved in most drug metabolism are well known. Drug metabolic enzymes are genetically polymorphic and phenotypes are determined by these genetic polymorphisms. Genetic polymorphisms of CYP2D6 (cytochrome P450 2D6), CYP2C19 (cytochrome P450 2C19) and CYP2C9 (cytochrome P450 2C9) are well known to have important consequences for the clinical effects of these substrate drugs. In particular, more than 50 gene variants of CYP2D6 isozymes are currently known, many of which are classified as pore metabolizer (PM) phenotypes with no drug metabolism by losing enzyme activity, but some gene variants (eg CYP2D6 * 9 , CYP2D6 * 10 , CYP2D6 * 17 ) may be shown in the form of decreased enzyme activity. In addition to these genetic polymorphisms, metabolic enzymes also have regulatory DNA elements that regulate their transcription (Gonzalez FJ., Molecular genetics of the P450-family. 1990; 45: 1-). 38) Abnormalities in transcriptional regulation may also affect the activity of enzymes. Genetic defects of the CYP2D6 isoenzyme cause various clinical effects due to various differences in its substrate drugs, such as cardiovascular and central nervous system drugs. For example, after administration of codeine, a precursor of morphine, its analgesic and respiratory inhibitory effects were not seen in the PM group, and in East Asians whose enzyme activity was relatively lower than that of Westerners. Appears relatively low. This difference is converted by the CYP2D6 isoenzyme into its active metabolite, morphine, because the effect is diminished by the reduction of morphine production in individuals who are genetically deficient or reduced. Similarly, substrate drugs of CYP2D6 isoenzyme such as tricyclic antidepressants, SSRI antidepressants, and many antipsychotic drugs show significant differences in blood drug levels after administration of the same dose of drug according to the individual's CYP2D6 phenotype and genotype. This may cause a difference in blood concentrations of more than 40 times.

On the other hand, HNF-4α is a transcriptional regulator belonging to the nuclear receptor group in which DNA-binding domains and ligand-bindings are preserved. HNF-4α is expressed in the liver, kidney, intestine, stomach and pancreas, and mutations in the HNF-4α gene are associated with disease on diabetes on the disease (1). Fajans SS, Bell GI, Polonsky KS., Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young.N Engl J Med 2001; 345: 971-980, Miquerol L, Lopez S, Cat¸ Tuliez M, Raymondjean M, Kahn A., Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas. J Biol chem. 1994; 269: 894-8951). The HNF-4α not only regulates transcription of genes required for cholesterol, fatty acid and glucose metabolism and liver differentiation (Jian G, Nepomuceno L, Hopkins k, Sladek FM., Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.Molecular and cell biology 1995 15: 5131-5143) (William C, Chrisopher ADS, Aileen WM, Roland W., Charaterization of the human cytochrome P4502D6 promoter.The Journal of Biological Chemistry. 1996 271 (41): 25269-25276, Ramiro J, Roque B, Maria J, Gomez-L, Jose VC., Cytochrome P450 regulation by hepatic nuclear factor 4 in human hepatocytes: A study using adenovirus-mediated antisense targeting.Hepatology 2001 33 (3): 668-675).

  HNF-4α is known to exhibit genetic polymorphism in MODY1 patients. However, a single base polymorphism at the splicing site involved in the exon encoding protein or the modification of the mRNA is known in normal non-patients. It has been reported that the first position cytosine is replaced by thymine, and as a result, the amino acid is replaced by isoleucine in threonine (Yamagata K, Furota H, Oda N., Mutation). in the hepatocyte nuclear factor-4 gene in maturity-onset diabetes o the young (MODY1) .Nature, 1996; 15: 5131-5143). However, there are no known examples of transcription-induced changes in drug metabolase according to mutation of HNF-4α gene in normal persons.

Therefore, the present inventors newly discovered monobasic polymorphism in the second exon region of HNF-4α during the study of monobasic polymorphism of HNF-4α in normal people, and the expression of CYP2D6, a representative drug metabolic enzyme, was significantly reduced due to the monobasic polymorphism. The present invention has been completed by identifying that it can be.

Accordingly, it is an object of the present invention to provide a method for detecting a polynucleotide comprising a single base polymorphic site of the HNF-4α gene.

Another object of the present invention is to provide a kit for diagnosing a disease caused by a deficiency of the CYP2D6 enzyme including a primer capable of amplifying the polynucleotide.

In order to achieve the above object, the present invention

(a) taking a biological sample from a human;

(b) extracting nucleic acids from the sample taken in step (a); And

(c) analyzing the sequence of the nucleic acid of step (b) to determine whether the 179th base of the HNF-4α gene is substituted with adenine (A) in guanine (G). Provided are methods of detecting nucleotides.

The present invention also provides a kit for diagnosing a disease caused by a deficiency of a CYP2D6 enzyme including a primer pair, a molecular weight marker, a DNA polymerase, and a buffer capable of amplifying a base sequence containing the 179th base of the HNF-4α gene. do.

Hereinafter, the present invention will be described in detail.

Polynucleotide containing a single base polymorphism of HNF-4α according to the present invention is a base containing a base in which the 179th base in the base sequence of HNF-4α of SEQ ID NO: 1 is substituted with adenine (A) in guanine (G) It is characterized by including the sequence. The polynucleotides of the invention also include their complements.

In one embodiment of the present invention, the single base polymorphism of the HNF-4α gene was analyzed in genomic DNA of 50 healthy Koreans. As a result, it was confirmed that the 179th base in the second exon region of the HNF-4α gene, ie, the 179th base in the nucleotide sequence of the HNF-4α gene of SEQ ID NO: 1, was changed from guanine to adenine (FIGS. 1 and 2). ). In addition, the single base polymorphism is examined to investigate whether or not it is located in the strand of HNF-4α and whether there is no other genetic variation in the same DNA strand or is due to a similar gene located in another part of the chromosome. DNA of the subject containing the mutant gene in which the polymorphism was found was templated, divided into 17 fragments, and PCR was performed to analyze the sequence of the amplified product. As a result, the test subject showed that one of the two strands of DNA had a variant and one strand had a wild type. The frequency of these mutations was 3% in Koreans. Single base polymorphism of the newly identified HNF-4α gene in the present invention causes the production of aspartic acid (D) instead of glycine (G) at the 60th amino acid position in the amino acid sequence of SEQ ID NO: 2 (Fig. 2). Therefore, the inventor named the single base polymorphism 'G60D mutation'.

Since the HNF-4α gene containing the mutation is not expressed by the normal HNF-4α protein, individuals with this mutation are likely to influence the activity of CYP2D6, a representative drug metabolic enzyme (Ramiro J, Roque B). , Maria J, Gomez-L, Jose VC., Cytochrome P450 regulation by hepatic nuclear factor 4 in human hepatocytes: A study using adenovirus-mediated antisense targeting.Hepatology 2001 33 (3): 668-675, William C, Chrisopher ADS, Aileen WM, Roland W., Charaterization of the human cytochrome P4502D6 promoter.The Journal of Biological Chemistry. 1996 271 (41): 25269-25276).

  Therefore, in another embodiment of the present invention, in order to investigate the effect of G60D mutation of HNF-4α on the activity of CYP2D6, the activity of the CYP2D6 promoter by the mutation of HNF-4α was analyzed. As a result of investigating the relative luciferase activity using the luciferase gene as a reporter gene, it was shown that the activity of the CYP2D6 promoter was significantly reduced by the G60D mutation of HNF-4α (FIG. 3).

In another embodiment of the present invention, the binding of the variant HNF-4α protein and the CYP2D6 promoter of the present invention was analyzed by an electrophoretic mobility shift assay (EMSA). As a result, the variant HNF-4α protein of the present invention was found not to bind to the CYP2D6 promoter (FIG. 4).

Polynucleotides of the invention may be operably linked to expression control sequences. “Operably linked” refers to the binding of one nucleic acid fragment to another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment. Also, “expression control sequence” refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences regulating termination of transcription and translation.

Polynucleotides according to the invention can be inserted into suitable expression vectors. As used herein, the term "expression vector" refers to a plasmid, virus or other medium known in the art to which a polynucleotide of the present invention may be inserted. Recombinant vectors comprising the polynucleotides according to the invention can be introduced into cells using methods known in the art. The cells may be eukaryotic cells such as yeast, plant cells or prokaryotic cells such as E. coli. By the known methods for introducing the expression vector according to the present invention into a host cell include, but are not limited to, Agrobacterium mediated transformation methods (Agrobacterium -mediated transformation), particle gun bombardment method (particle gun bombardment), silicon Silicon carbide whiskers, sonication, electroporation, and precipitation by polyethylene glycol (PEG) may be used.

The monobasic polymorphism of HNF-4α according to the present invention can be detected by a method comprising the following steps.

(a) taking a biological sample from a human;

(b) extracting nucleic acids from the sample taken in step (a); And

(c) analyzing the nucleic acid sequence of step (b) to determine whether the 179th base of HNF-4α is substituted with adenine for guanine.

In the step (a), the biological sample may be blood, skin cells, mucosal cells and hair of the subject. Preferably blood.

In step (b), the nucleic acid may be DNA or RNA, preferably DNA. The method of extracting nucleic acids from the sample collected in step (a) is not particularly limited, and techniques known in the art or commercially available kits for extraction may be used. For example, kits for DNA or RNA extraction can be purchased from Qiagen, Inc. (California, USA) and Stratagene (California, USA). In the case of extracting and using RNA from the above, cDNA is prepared by reverse transcription.

The method of analyzing the nucleic acid sequence in step (c) to determine whether the 179th base of HNF-4α is substituted with guanine for adenine is not particularly limited, and methods known in the art may be used. For example, an autobase sequencer or pyrosequencing may be performed as a method of directly determining the sequence portion. The pyro sequencing is a well-known SNP analysis method that is also used for DNA sequencing is a method of detecting the expression of light of PPi (inorganic pyrophosphate) emitted during the polymerization of DNA. In addition, PCR-RFLP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), PCR-SSO and all hybrid specific oligonucleotide (ASO) combined with dot hybridization Hybridization method, TaqMan-PCR method, MALDI-TOF / MS method, rolling circle amplification method, method using DNA chip or microarray, invader method, primer extension method, Southern blot hybridization method, dot hybridization method, etc. Known methods can be used. Furthermore, the above various kinds of sequencing methods may be used in combination. It can also be detected indirectly by analyzing the activity of the CYP2D6 promoter. In addition, it can be detected through various monobasic polymorphism analysis methods known in the art.

In addition, detection of a single nucleotide polymorphism of HNF-4α according to the present invention can amplify a continuous base sequence containing the 179th base of HNF-4α of SEQ ID NO: 1 using the nucleic acid of step (b) as a template. PCR amplification with a primer may be further performed. Preferably, the primer may be one capable of amplifying a nucleotide sequence including the second exon region of HNF-4α. The primers can be designed according to methods known in the art based on the sequence of known HNF-4α (SEQ ID NO: 1).

The monobasic polymorphism of HNF-4α according to the present invention causes the production of aspartic acid expression code instead of the normal glycine expression code at the 60th amino acid position of SEQ ID NO: 2 as described above (see FIG. 2). The variant HNF-4α protein expressed from the polynucleotide of the present invention does not normally bind to the promoter of CYP2D6, unlike the wild type HNF-4α protein. As a result, the CYP2D6 promoter does not work properly, and as a result, the expression of CYP2D6, which is regulated by the promoter, is also poorly achieved. Therefore, individuals with G60D mutation of HNF-4α identified in the present invention are deficient in CYP2D6 enzyme, a drug metabolic enzyme, and thus cannot perform normal drug metabolism. Therefore, the single-base polymorphism of HNF-4α newly discovered in the present invention may induce an adverse drug reaction caused by the deficiency of the CYP2D6 enzyme. Therefore, the single-base polymorphism of the present invention is useful as a labeling factor correlated with abnormal function of drug metabolism. Can be used. That is, the detection of a single nucleotide polymorphism of HNF-4α of the present invention can predict and diagnose an individual difference in CYP2D6 enzyme activity or an adverse reaction due to a deficiency of CYP2D6 enzyme. It is also known that HNF-4α is necessary for the preservation of normal liver function and is associated with the development of diabetes mellitus (Gaedigk A, Blum M, Gaedigk R, Eichelbaum M, Meyer UA., Deletion of the entire cytocheome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizer of the debrisoquin / sparteine polymorphism.American Journal of Human Genetics. 1991 48 (5): 943-950).

In addition, the present invention is a kit for diagnosing abnormal symptoms caused by a deficiency of the CYP2D6 enzyme, characterized in that it comprises a primer capable of specifically amplifying a region containing a single nucleotide polymorphism site of HNF-4α according to the present invention. To provide. Preferably, the primer may be one capable of amplifying the second exon region of HNF-4α. In addition, primers designed to form specific restriction enzyme sites may be used when the sequence is to be analyzed by treating the amplified PCR product with restriction enzymes. Although the base length of an appropriate primer is not specifically limited, For example, about 15-30bp can be used. Kits according to the invention may further comprise a molecular weight marker, DNA polymerase and buffer in addition to the primer.

In addition, since HNF-4α is closely related to diabetes, the single nucleotide polymorphism of HNF-4α of the present invention may be useful for elucidating the mechanism of diabetes and / or for predicting the possibility of diabetes. In addition, the single-base polymorphism of the HNF-4α of the present invention can be very useful for the development of personalized drugs through the study of individual drug response due to genetic differences.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1>

HNF-4 Monobasic polymorphism analysis of α gene

Blood was collected from 50 healthy Korean subjects and genomic DNA was isolated using a genomic DNA isolation kit (Qiagen). The entire nucleotide sequence of the HNF-4α gene in the genomic DNA was analyzed using an automatic base sequence analyzer (ABI 3700, ABI). As a result, a single base polymorphism was found in the HNF-4α gene of three subjects. That is, in the base sequence of HNF-4α of SEQ ID NO: 1, it was confirmed that guanine (G), which is the 91st base of the second exon region, was mutated to adenine (A).

The inventors then examined whether the single nucleotide polymorphisms found were located on the strand of HNF-4α, whether there were no other genetic mutations in the same DNA strand, and whether they were from similar genes located in different parts of the chromosome. The base sequence was analyzed by cloning by PCR amplification using a single strand of DNA as a template from the DNA of the subject including the mutant gene in which the single nucleotide polymorphism was found.

Since the HNF-4α gene includes 10 exons and has an intron therebetween, the total gene length is more than 30,000 bases, and the present inventors performed PCR by dividing the HNF-4α gene into 17 fragments. The primer pairs used for each PCR, the position and size of the PCR products amplified by the primer pairs and the PCR reaction conditions are shown in Tables 1 to 3, respectively.

Primer Pairs Used for PCR PCR products Primer Name Sequence (5 '→ 3') SEQ ID NO: HNF4A-P1 HNF4A-P1F AAC TTC CAG TCC ATT CTG CTC C 3 HNF4A-P1R AGT GCC CTC TCT GCC TTC C 4 HNF4A-P2 HNF4A-P2F CTG TGA ATG CAC TGG CGA TA 5 HNF4A-P2R CCA GAT GCC AAT CTT CTG GAT C 6 HNF4A-P3 HNF4A-P3F ACC CAC CCA TTT GCT CAC 7 HNF4A-P3R AAG TCC TGG TTC CTG GTT CA 8 HNF4A-P4 HNF4A-P4F GTC TCA TAG TGT GGG TTG CAG 9 HNF4A-P4R TGA GTG TGT GGG GGA ATT GAT G 10 HNF4A-E1 HNF4A-E1F CAA GAC TCC CAG CAG ATC TTC 11 HNF4A-E1R AAG ATC TGC TCC TGG ACC CA 12 HNF4A-E2 HNF4A-E2F AAG GCT CCC TTA GAT GCC T 13 HNF4A-E2R TCA GGG AGA AGA CAG ACC TTG 14 HNF4A-E3 HNF4A-E3F CTG TCC TAA GAG GAG GAA GTT 15 HNF4A-E3R GAG CTG TGG GTG CAG GTG 16 HNF4A-E4 HNF4A-E4F CTG CTC CCA CTC CTC ATC AGT 17 HNF4A-E4R ATG TGA AAC CGG ACT CAG TGT C 18 HNF4A-E5 HNF4A-E5F CTG TGC AGG GGA CAG AGA 19 HNF4A-E5R AGC CAG TCC ACG GCT ATA TC 20 HNF4A-E6 HNF4A-E6F AGT TGG CTA CGG GCA GCC 21 HNF4A-E6R TGA GTG CCA TGC TAG GCA T 22 HNF4A-E7 HNF4A-E7F GGC ACC AGC TAT CTT GCC A 23 HNF4A-E7R AGG AGA AGT CTG GCA GAG C 24 HNF4A-E8 HNF4A-E8F ATG CTC CAG CTG GAC CCT G 25 HNF4A-E8R GTG TGA GGC CTG TCT CCT G 26 HNF4A-E9 HNF4A-E9F AGG TCT GCA TCC CAG ACT C 27 HNF4A-E9R TCT CCT GCC TCC TGT CTT AAG 28 HNF4A-E10 (1) HNF4A-E10F (1) CAT TTA CTC CCA CAA AGG CTG 29 HNF4A-E10R (1) GTC ATC TCC AGG CGG CTG T 30 HNF4A-E10 (2) HNF4A-E10F (2) CCT TCA CCT TCA CCT TCA TCC A 31 HNF4A-E10F (2) AAG TCC CTG AAG AAG GAG GAT G 32 HNF4A-E10 (3) HNF4A-E10F (3) GAA GGC TGT GGT GAG GGA A 33 HNF4A-E10R (3) GAG ATG ATG CAT GTC AGA TGC C 34 HNF4A-E10 (4) HNF4A-E10F (4) CCT CAC ATC AGA GTG ACA TCC A 35 HNF4A-E10F (4) TGA CTT GGG GAG TGA AGA GAG A 36

Location and Size of PCR Products PCR products location size HNF4A-P1 -691 ~ -669 575 -135--116 HNF4A-P2 -1159 ~ -1138 563 -618 ~ -596 HNF4A-P3 -1716 ~ -1698 540 -1196 ~ -1176 HNF4A-P4 -2194--2173 629 -1587--1565 HNF4A-E1 -174 ~ -153 447 IVS1 + 49 ~ IVS1 + 69 HNF4A-E2 IVS2-66 ~ IVS2-47 317 IVS2 + 55 ~ IVS2 + 76 HNF4A-E3 IVS3-58-IVS3-37 208 IVS3 + 37-IVS3 + 55 HNF4A-E4 IVS4-87-IVS4-66 345 IVS4 + 129-IVS4 + 151 HNF4A-E5 IVS5-70 ~ IVS5-52 319 IVS5 + 73 ~ IVS5 + 93 HNF4A-E6 IVS6-63 ~ IVS6-45 211 IVS6 + 41 ~ IVS6 + 60 HNF4A-E7 IVS7-95-IVS7-76 316 IVS7 + 45 ~ IVS7 + 64 HNF4A-E8 IVS8-52 ~ IVS8-33 353 IVS8 + 45 ~ IVS8 + 64 HNF4A-E9 IVS9-49 ~ IVS9-30 268 IVS9 + 45 ~ IVS9 + 66 HNF4A-E10 (1) IVS10-61 ~ IVS10 + 40 531 Exon10 + 451 ~ Exon10 + 470 HNF4A-E10 (2) Exon10 + 401 ~ Exon10 + 423 443 Exon10 + 822 ~ Exon10 + 844 HNF4A-E10 (3) Exon10 + 777 ~ Exon10 + 796 650 Exon10 + 1405 ~ Exon10 + 1427 HNF4A-E10 (4) Exon10 + 1323 ~ Exon10 + 1345 584 IVS10 + 18 ~ IVS10 + 39

℃

PCR reaction conditions PCR products Reaction condition HNF4A-P1 94 ° C 5 minutes → (94 ° C 30 seconds, 58.3 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-P2 94 ° C 5 minutes → (94 ° C 30 seconds, 62 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-P3 94 ° C 5 minutes → (94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-P4 94 ° C 5 minutes → (94 ° C 30 seconds, 53 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-E1 94 ° C 5 minutes → (94 ° C 30 seconds, 62 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E2 94 ° C 5 minutes → (94 ° C 30 seconds, 56.3 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E3 94 ° C 5 minutes → (94 ° C 30 seconds, 58.3 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E4 94 ℃ 5 minutes → (94 ℃ 30 seconds, 49.3 ℃ 30 seconds, 72 ℃ 30 seconds) 35 cycles → 72 ℃ 7 minutes HNF4A-E5 94 ° C 5 minutes → (94 ° C 30 seconds, 55.3 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E6 94 ° C 5 minutes → (94 ° C 30 seconds, 62 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E7 94 ° C 5 minutes → (94 ° C 30 seconds, 56.3 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E8 94 ° C 5 minutes → (94 ° C 30 seconds, 62.7 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E9 94 ° C 5 minutes → (94 ° C 30 seconds, 53 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E10 (1) 94 ° C 5 minutes → (94 ° C 30 seconds, 52.4 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-E10 (2) 94 ° C 5 minutes → (94 ° C 30 seconds, 52.4 ° C 30 seconds, 72 ° C 30 seconds) 35 cycles → 72 ° C 7 minutes HNF4A-E10 (3) 94 ° C 5 minutes → (94 ° C 30 seconds, 56.3 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes HNF4A-E10 (4) 94 ° C 5 minutes → (94 ° C 30 seconds, 56.3 ° C 30 seconds, 72 ° C 1 minute) 35 cycles → 72 ° C 7 minutes

Thereafter, the nucleotide sequence of the amplified product was confirmed by an automatic base sequence analyzer. The sequencing primer used for this is shown in Table 4 below.

Sequencing Primers for Each PCR Product PCR products Sequencing primers HNF4A-P1 HNF4A-P1F, P1R HNF4A-P2 HNF4A-P2F, P2R HNF4A-P3 HNF4A-P3F, P3R HNF4A-P4 HNF4A-P4F, P4R HNF4A-E1 HNF4A-E1F, E1R HNF4A-E2 HNF4A-E2F, E2R HNF4A-E3 HNF4A-E3F, E3R HNF4A-E4 HNF4A-E4F, E4R HNF4A-E5 HNF4A-E5F, E5R HNF4A-E6 HNF4A-E6F, E6R HNF4A-E7 HNF4A-E7F, E7R HNF4A-E8 HNF4A-E8F, E8R HNF4A-E9 HNF4A-E9F, E9R HNF4A-E10 (1) HNF4A-E10F (1), E10R (1) HNF4A-E10 (2) HNF4A-E10F (2), E10R (2) HNF4A-E10 (3) HNF4A-E10F (3), E10R (3) HNF4A-E10 (4) HNF4A-E10F (4), E10R (4)

As a result of the sequencing analysis, guanine, which is the base of the 91st position in the second exon of the HNF-4α gene, was transformed into adenine in the same manner as in the first experiment (FIG. 1). One of the strands of DNA is mutant and one strand is wild type. Thus, if guanine is replaced with adenine at the 91th position of the second exon of the HNF-4α gene, that is, at the 179th position of the base sequence of the HNF-4α gene of SEQ ID NO: 1, the 60th amino acid position of the amino acid sequence of SEQ ID NO: 2 Mutant HNF-4α protein is produced in which glycine (G) is substituted with aspartic acid (D). Therefore, the inventors named the mutation 'G60D mutation'.

<Example 2>

Activity Analysis of CYP2D6 Promoter by G60D Mutation of HNF-4α Gene

Since the individual having the G60D mutation of the HNF-4α gene identified in Example 1 is highly likely to affect the promoter activity of CYP2D6, the present inventors measured the activity of the CYP2D6 promoter due to the mutation. In order to prepare a vector in which the luciferase gene is linked to the CYP2D6 promoter, blood was first collected from blood of a normal subject, and genomic DNA was isolated using a genomic DNA separation kit (Qiagen). Using the isolated genomic DNA as a template and amplifying the -1849 to +1 region of the CYP2D6 promoter by PCR using the CYP2D6-1.8kb-F primer (SEQ ID NO: 37) and the CYP2D6-1.8kb-R primer (SEQ ID NO: 38). It was. PCR reaction conditions were 94 degreeC 5 minutes ((94 degreeC 30 second, 62 degreeC 30 second, 72 degreeC 2 minutes) 35 cycles → 72 degreeC 5 minutes. The amplified PCR product was digested with Xho I and Hin dIII and cloned into pGL2 vector (Promega). The prepared vector was named 'pGL2-CYP2D6'.

On the other hand, the vector containing the mutated HNF-4α gene is pRC / CMV2-hHNF-4-WT (Terumasa T, Yoshihiko k, Masatsugu U., Human hypoxic signal transduction through a signature motif in hepatocyte nuclear) factor 4. Journal of Biochemistry. 2002 132: 37-44) was used to prepare a site-directed mutagenesis kit (Stratagene), which was prepared using 'pRC / CMV2-hHNF-4-G60D'. It was named. As a control, a pRC / CMV2-hHNF-4-WT vector expressing a normal HNF-4α gene was used.

The prepared vector was introduced into 5 × 10 ⁵ 293F cells (Invitorgen) using Lipofectamine ™ 2000 (Invitrogen). Luciferase activity was analyzed using a Luciferase reporter gene assay reagent (Roche).

In addition, pCMV-β-gal vector (Promega Co., Ltd. was introduced into the cell at the same time. The activity of β-galactosidase was determined using CPRG (chlorophenol red-β-D-galactopyranoside, Roche Co., Ltd.) as a substrate. Was analyzed by measuring the absorbance at 570 nm.

As a result, as shown in Figure 3, the G60D mutation of the HNF-4α gene was confirmed that the activity of the CYP2D6 promoter is reduced by up to 6 times. This indicates that the expression of the CYP2D6 gene is inhibited by the mutation of the HNF-4α gene.

<Example 3>

Coupling Analysis of Mutant HNF-4α Proteins with CYP2D6 Promoter

To investigate the binding of the CYP2D6 promoter by G60D mutation of the HNF-4α gene, an electrophoretic mobility shift assay (EMSA) analysis was performed. The wild type HNF-4α protein and its G60D variant protein were produced by the manufacturer's recommended method using Promega's TNT Quick Coupled Transcription / Translation Systems. In addition, the G60D variant gene of HNF-4α was amplified using HNF4A-F-BamHI primer (SEQ ID NO: 39) and HNF4A-R-EcoRI primer (SEQ ID NO: 40). At this time, PCR reaction conditions were 94 degreeC 5 minutes → (94 degreeC 30 second, 60 degreeC 45 second, 72 degreeC 3 minutes) 30 cycles → 72 degreeC 7 minutes. Then, the amplified mutant gene was inserted into the pcDNA3,1 (+) (Invitrogen) vector and expressed as a protein.

-³²P]ATP로 표지하였다. ³²P로 표지된 올리고뉴클레오티드탐침을 약 10 ㎍의 야생형 HNF-4α 단백질 및 이의 G60D 변이형 단백질과 각각 결합시켰다. 결합 반응액은 5X binding buffer(20% glycerol, 100 mM Tris (pH 8.0), 300 mM KCl, 25mM MgCl2) 4 ㎕, Didc 2 ㎕, 야생형 HNF-4α 및 G60D 변이형 단백질 2 ㎕, D.W 9 ㎕를 혼합하여 4℃에서 30분간 반응시킨 후 올리고뉴클레오티드탐침 2 ㎕를 첨가하여 실온에서 30분 동안 반응시켜 결합 하였다. 경쟁적 반응 실험을 위하여 상기 ³²P로 표지된 올리고뉴클레오티드 탐침과 반응시키기 전에 competitor로서 1-10 pmol의 미표지 올리고뉴클레오티드 탐침과 30분간 미리 반응시켰다. 음성 대조군으로는 야생형 HNF-4α 및 G60D 변이형 단백질을 제외한 나머지 반응물을 결합하여 사용하였다. DNA-단백질 결합 반응은 4% 폴리아크릴아미드 겔에서 분석한 후, 분석결과는 자동방사선 사진으로 가시화하였다. An oligonucleotide (SEQ ID NO: 41) at the -56 to 36 position containing the HNF-4α binding site was used as a probe CYP2D6 promoter sequence for EMSA analysis. The oligonucleotide probe is T4-polynucleotide kinase (polynucleotide kinase) using [

Labeled with ³² P] ATP. Oligonucleotide probes labeled with ³² P were bound with about 10 μg of wild type HNF-4α protein and its G60D variant protein, respectively. The binding reaction solution was prepared with 4 μl of 5X binding buffer (20% glycerol, 100 mM Tris (pH 8.0), 300 mM KCl, 25 mM MgCl 2), 2 μl Didc, 2 μl wild type HNF-4α and G60D variant protein, 9 μl DW. After mixing and reacting at 4 ° C. for 30 minutes, 2 μl of an oligonucleotide probe was added thereto, followed by reaction at room temperature for 30 minutes for binding. Before the reaction with the ³² P-labeled oligonucleotide probe for a competitive reaction experiment, it was reacted with the unlabeled oligonucleotide probe of 1-10 pmol for 30 minutes as a competitor. The negative control was used in combination with the rest of the reactants except for the wild type HNF-4α and G60D variant proteins. DNA-protein binding reactions were analyzed on 4% polyacrylamide gels, and the results were visualized by autoradiography.

As a result, as shown in FIG. 4, the G60D variant protein of HNF-4α did not bind to the CYP2D6 promoter.

As described above, the monobasic polymorphism of HNF-4α of the present invention may be usefully used as a labeling factor correlated with abnormal function of drug metabolism. That is, it is possible to predict and diagnose abnormal symptoms due to individual differences in CYP2D6 enzymatic activity or deficiency of CYP2D6 enzyme through detection of HNF-4α monobasic polymorphism of the present invention. In addition, it can be very useful for the development of personalized drugs through individual drug response due to genetic differences. In addition, since HNF-4α is closely related to diabetes, the single nucleotide polymorphism of HNF-4α of the present invention may be useful for elucidating the mechanism of diabetes and / or for predicting the possibility of diabetes.

<110> INJE UNIVERSITY <120> Single nucleotide polymorphism of HNF-4 alpha gene and use          about <130> DPP050118KR <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 1398 <212> DNA <213> Homo sapiens <400> 1 atggacatgg ccgactacag tgctgcactg gacccagcct acaccaccct ggaatttgag 60 aatgtgcagg tgttgacgat gggcaatgac acgtccccat cagaaggcac caacctcaac 120 gcgcccaaca gcctgggtgt cagcgccctg tgtgccatct gcggggaccg ggccacggac 180 aaacactacg gtgcctcgag ctgtgacggc tgcaagggct tcttccggag gagcgtgcgg 240 aagaaccaca tgtactcctg cagatttagc cggcagtgcg tggtggacaa agacaagagg 300 aaccagtgcc gctactgcag gctcaagaaa tgcttccggg ctggcatgaa gaaggaagcc 360 gtccagaatg agcgggaccg gatcagcact cgaaggtcaa gctatgagga cagcagcctg 420 ccctccatca atgcgctcct gcaggcggag gtcctgtccc gacagatcac ctcccccgtc 480 tccgggatca acggcgacat tcgggcgaag aagattgcca gcatcgcaga tgtgtgtgag 540 tccatgaagg agcagctgct ggttctcgtt gagtgggcca agtacatccc agctttctgc 600 gagctccccc tggacgacca ggtggccctg ctcagagccc atgctggcga gcacctgctg 660 ctcggagcca ccaagagatc catggtgttc aaggacgtgc tgctcctagg caatgactac 720 attgtccctc ggcactgccc ggagctggcg gagatgagcc gggtgtccat acgcatcctt 780 gacgagctgg tgctgccctt ccaggagctg cagatcgatg acaatgagta tgcctacctc 840 aaagccatca tcttctttga cccagatgcc aaggggctga gcgatccagg gaagatcaag 900 cggctgcgtt cccaggtgca ggtgagcttg gaggactaca tcaacgaccg ccagtatgac 960 tcgcgtggcc gctttggaga gctgctgctg ctgctgccca ccttgcagag catcacctgg 1020 cagatgatcg agcagatcca gttcatcaag ctcttcggca tggccaagat tgacaacctg 1080 ttgcaggaga tgctgctggg agggtccccc agcgatgcac cccatgccca ccaccccctg 1140 caccctcacc tgatgcagga acatatggga accaacgtca tcgttgccaa cacaatgccc 1200 actcacctca gcaacggaca gatgtgtgag tggccccgac ccaggggaca ggcagccacc 1260 cctgagaccc cacagccctc accgccaggt gcgtcagggt ctgagcccta taagctcctg 1320 ccgggagccg tcgccacaat cgtcaagccc ctctctgcca tcccccagcc gaccatcacc 1380 aagcaggaag ttatctag 1398 <210> 2 <211> 465 <212> PRT <213> Homo sapiens <400> 2 Met Asp Met Ala Asp Tyr Ser Ala Ala Leu Asp Pro Ala Tyr Thr Thr   1 5 10 15 Leu Glu Phe Glu Asn Val Gln Val Leu Thr Met Gly Asn Asp Thr Ser              20 25 30 Pro Ser Glu Gly Thr Asn Leu Asn Ala Pro Asn Ser Leu Gly Val Ser          35 40 45 Ala Leu Cys Ala Ile Cys Gly Asp Arg Ala Thr Asp Lys His Tyr Gly      50 55 60 Ala Ser Ser Cys Asp Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Arg  65 70 75 80 Lys Asn His Met Tyr Ser Cys Arg Phe Ser Arg Gln Cys Val Val Asp                  85 90 95 Lys Asp Lys Arg Asn Gln Cys Arg Tyr Cys Arg Leu Lys Lys Cys Phe             100 105 110 Arg Ala Gly Met Lys Lys Glu Ala Val Gln Asn Glu Arg Asp Arg Ile         115 120 125 Ser Thr Arg Arg Ser Ser Tyr Glu Asp Ser Ser Leu Pro Ser Ile Asn     130 135 140 Ala Leu Leu Gln Ala Glu Val Leu Ser Arg Gln Ile Thr Ser Pro Val 145 150 155 160 Ser Gly Ile Asn Gly Asp Ile Arg Ala Lys Lys Ile Ala Ser Ile Ala                 165 170 175 Asp Val Cys Glu Ser Met Lys Glu Gln Leu Leu Val Leu Val Glu Trp             180 185 190 Ala Lys Tyr Ile Pro Ala Phe Cys Glu Leu Pro Leu Asp Asp Gln Val         195 200 205 Ala Leu Leu Arg Ala His Ala Gly Glu His Leu Leu Leu Gly Ala Thr     210 215 220 Lys Arg Ser Met Val Phe Lys Asp Val Leu Leu Leu Gly Asn Asp Tyr 225 230 235 240 Ile Val Pro Arg His Cys Pro Glu Leu Ala Glu Met Ser Arg Val Ser                 245 250 255 Ile Arg Ile Leu Asp Glu Leu Val Leu Pro Phe Gln Glu Leu Gln Ile             260 265 270 Asp Asp Asn Glu Tyr Ala Tyr Leu Lys Ala Ile Ile Phe Phe Asp Pro         275 280 285 Asp Ala Lys Gly Leu Ser Asp Pro Gly Lys Ile Lys Arg Leu Arg Ser     290 295 300 Gln Val Gln Val Ser Leu Glu Asp Tyr Ile Asn Asp Arg Gln Tyr Asp 305 310 315 320 Ser Arg Gly Arg Phe Gly Glu Leu Leu Leu Leu Leu Pro Thr Leu Gln                 325 330 335 Ser Ile Thr Trp Gln Met Ile Glu Gln Ile Gln Phe Ile Lys Leu Phe             340 345 350 Gly Met Ala Lys Ile Asp Asn Leu Leu Gln Glu Met Leu Leu Gly Gly         355 360 365 Ser Pro Ser Asp Ala Pro His Ala His His Pro Leu His Pro His Leu     370 375 380 Met Gln Glu His Met Gly Thr Asn Val Ile Val Ala Asn Thr Met Pro 385 390 395 400 Thr His Leu Ser Asn Gly Gln Met Cys Glu Trp Pro Arg Pro Arg Gly                 405 410 415 Gln Ala Ala Thr Pro Glu Thr Pro Gln Pro Ser Pro Pro Gly Ala Ser             420 425 430 Gly Ser Glu Pro Tyr Lys Leu Leu Pro Gly Ala Val Ala Thr Ile Val         435 440 445 Lys Pro Leu Ser Ala Ile Pro Gln Pro Thr Ile Thr Lys Gln Glu Val     450 455 460 Ile 465 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P1F primer <400> 3 aacttccagt ccattctgct cc 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4-P1R primer <400> 4 agtgccctct ctgccttcc 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P2F primer <400> 5 ctgtgaatgc actggcgata 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P2R primer <400> 6 ccagatgcca atcttctgga tc 22 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P3F primer <400> 7 acccacccat ttgctcac 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P3R primer <400> 8 aagtcctggt tcctggttca 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P4F primer <400> 9 gtctcatagt gtgggttgca g 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-P4R primer <400> 10 tgagtgtgtg ggggaattga tg 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E1F primer <400> 11 caagactccc agcagatctt c 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E1R primer <400> 12 aagatctgct cctggaccca 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> NHF4A-E2F primer <400> 13 aaggctccct tagatgcct 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E2R primer <400> 14 tcagggagaa gacagacctt g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E3F primer <400> 15 ctgtcctaag aggaggaagt t 21 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E3R primer <400> 16 gagctgtggg tgcaggtg 18 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E4F primer <400> 17 ctgctcccac tcctcatcag t 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E4R primer <400> 18 atgtgaaacc ggactcagtg tc 22 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E5F primer <400> 19 ctgtgcaggg gacagaga 18 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E5R primer <400> 20 agccagtcca cggctatatc 20 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E6F primer <400> 21 agttggctac gggcagcc 18 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E6R primer <400> 22 tgagtgccat gctaggcat 19 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E7F primer <400> 23 ggcaccagct atcttgcca 19 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E7R primer <400> 24 aggagaagtc tggcagagc 19 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E8F primer <400> 25 atgctccagc tggaccctg 19 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E8R primer <400> 26 gtgtgaggcc tgtctcctg 19 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E9F primer <400> 27 aggtctgcat cccagactc 19 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E9R primer <400> 28 tctcctgcct cctgtcttaa g 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (1) primer <400> 29 catttactcc cacaaaggct g 21 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10R (1) primer <400> 30 gtcatctcca ggcggctgt 19 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (2) primer <400> 31 ccttcacctt caccttcatc ca 22 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (2) primer <400> 32 aagtccctga agaaggagga tg 22 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (3) primer <400> 33 gaaggctgtg gtgagggaa 19 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10R (3) primer <400> 34 gagatgatgc atgtcagatg cc 22 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (4) primer <400> 35 cctcacatca gagtgacatc ca 22 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-E10F (4) primer <400> 36 tgacttgggg agtgaagaga ga 22 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CYP2D6-1.8kb-F primer <400> 37 gatccctcga gcactggctc caagcatggc ag 32 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CYP2D6-1.8kb-R primer <400> 38 gatccctcga gcactggctc caagcatggc ag 32 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-F-BamHI primer <400> 39 agtaggatcc atggacatgg ccgactacag 30 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> HNF4A-R-EcoRI primer <400> 40 gcatgaattc ctagataact tcctgcttgg 30 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide probe for CYP2D6 promoter <400> 41 agcagagggc aaaggccatc atcag 25  

Claims (6)

(a) taking a biological sample from a human; (b) extracting nucleic acids from the sample taken in step (a); And (c) analyzing the sequence of the nucleic acid of step (b) to determine whether the 179th base of the HNF-4α gene is substituted with adenine (A) in guanine (G). Method for detecting polynucleotide of claim. The method of claim 1, wherein the biological sample of step (a) is selected from the group consisting of blood, skin cells, mucosal cells and hair. The method of claim 1, wherein in step (c), base substitution is performed by automatic sequencing, pyrosequencing, PCR-RFLP (restriction fragment length polymorphism), and PCR-SSCP (single strand conformation polymorphism). ), PCR-SSO (specific sequence oligonucleotide), ASO (allele specific oligonucleotide) hybridization method, TaqMan-PCR method, MALDI-TOF / MS method, rolling circle amplification method, DNA chip or microarray method, Invader method, primer extension method, Southern blot hybridization method, dot hybridization DNA sequencing, PCR-SSCP analysis, RFLP analysis and allyl specific hybridization method characterized in that the analysis using any one method selected from the group How to. According to claim 1, wherein the nucleic acid of the step (a) as a template characterized in that it further comprises the step of PCR amplification with a primer capable of amplifying the base sequence containing the 179th base of the HNF-4α gene Way. A kit for diagnosing a disease caused by a deficiency of a CYP2D6 enzyme comprising a primer pair, a molecular weight marker, a DNA polymerase and a buffer capable of amplifying a contiguous sequence containing the 179th base of the HNF-4α gene. The kit of claim 5, wherein the primer pair is capable of amplifying a nucleotide sequence including the second exon region of the HNF-4α gene.

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