JP2006101808A - Method for examining variation of ugtia4 nucleic acid sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examining the genetic variation of exon 1 domain of nucleic acid sequence of UGT1A4, a method for judging, predicting or examining medicine metabolism by using the examination method and a method for examining jaundice, cancer, coronary arteries disease or osteoporosis. <P>SOLUTION: New variation to cause amino acid substitution in UGT1A4 enzyme molecule is found by searching variation of exon 1 domain of nucleic acid sequence of UGT1A4. The examination of the variation finds that UGT1A4 activity is predicted and the variation deeply participates in medicine metabolism, jaundice, cancer, coronary arteries disease or osteoporosis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention is used in the field of clinical tests, and more particularly to a method for testing nucleic acid sequences of enzymes involved in glucuronidation and a kit therefor.

Glucuronidation catalyzed by UDP-glucuronyltransferase (UGT) plays an important role in the metabolism and detoxification of various drugs and poisons including bilirubin. The human UGT1 gene group is composed of at least 13 different exons 1 and common exons 2 to 5, and one exon 1 is spliced to common exons 2 to 5 (Non-patent Document 1). Each UGT has a broad substrate specificity and a unique distribution in living tissue.

UGT1A4 is expressed in the liver and stomach and is known to glucuronide-conjugate plant steroids such as primary amine, tertiary amine, beta naphthylamine, 4-aminobiphenyl, benzidine, androgen, progestin, and sapogenin. (Non-Patent Document 2, etc.). Recently, it has been reported that a functional polymorphism of UGT1A4 exists in patients with hepatocellular carcinoma (Non-patent Document 3).
J. Biol. Chem. (1992) 267, 1043-1047 Drug Metab. Dispos. (1998) 26, 507-512 Hepatorogy (2004) 39, 970-977

  An object of the present invention is to provide a method for testing gene mutations in the exon 1 region of UGT1A4 nucleic acid sequence and introns downstream thereof, and to determine, predict or test drug metabolism using this test method, jaundice, cancer, It is to provide a method for examining coronary artery disease or osteoporosis.

The present inventors searched for mutations in the exon 1 region of the nucleic acid sequence of UGT1A4 and the intron region downstream thereof, and found new mutations that cause amino acid substitutions in the UGT1A4 enzyme molecule. The present invention was completed by discovering that the prediction is possible and that it is deeply involved in drug metabolism, jaundice, cancer, coronary artery disease or osteoporosis.

The present invention has the following configuration.
1. A method for examining a nucleic acid sequence of UDP-glucuronosyltransferase (UGT1A4), which comprises examining the mutation from t to g (L48V) at position 142 in the sequence represented by SEQ ID NO: 1. Method for testing nucleic acid sequence of
2. A method for examining or predicting the function and enzyme activity of the UGT1A4 enzyme molecule by examining the nucleic acid sequence of UGT1A4 according to 1 above.
3. A method for examining drug metabolic capacity, jaundice, cancer, coronary artery disease or osteoporosis according to the method described in 2 above.
4. The inspection method according to any one of 1 to 3, wherein any one or at least two of the oligonucleotide primers described in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 9 are used.
5. An inspection apparatus, reagent, or kit used for the inspection method according to any one of 1 to 4 above.
6. A testing device, reagent or kit incorporating at least one oligonucleotide primer used in the testing method according to any one of 4 or 5 above.

The present invention provides a method for testing a new mutation of UGT1A4 that has not been known so far, and by using this test method, it is possible to test and predict drug metabolism, cancer, jaundice, coronary artery disease, and osteoporosis. It has a remarkable effect of becoming.

(Target of inspection)
Test subjects according to the present invention include drug metabolic ability, jaundice, cancer, coronary artery disease or osteoporosis.
In addition to the metabolic pathway by cytochrome P450, the metabolic pathway by glucuronidation by UGT1 is also important for drug metabolism, and by using the test method of the present invention, the efficacy and side effects of drugs metabolized by glucuronidation can be predicted. It becomes possible. Examples of the target drug that can be examined by the method of the present invention include primary amine, tertiary amine, beta naphthylamine, 4-aminobiphenyl, benzidine, androgen, progestin, and clozapine.

UGT1 is essential for the excretion of bilirubin from the liver, and the present invention is effective for examining the cause of jaundice. The present invention is also useful as a method for examining the association with various cancers including breast cancer and colorectal cancer, and is also a useful testing method for understanding and predicting the onset of coronary artery disease and osteoporosis. .

(Inspection sample)
The genomic DNA of the UGT1A4 nucleic acid sequence to be examined can be obtained without any particular limitation, such as organs, tissues such as liver, kidney, leukocytes, and hair. One of the methods for obtaining genomic DNA from leukocytes is a commercially available kit, for example, Qiagen's Quiagen Blood DNA kit. Using the obtained genomic DNA, genomic DNA containing the region described in SEQ ID NO: 1 is converted into, for example, 5′-TTTGTCTTCCAATTACATGC-3 ′ described in SEQ ID NO: 2 and 5′-AGATATGGAAGCACTTGTAAG-3 described in SEQ ID NO: 3. Amplification is performed by polymerase chain reaction (PCR) using the primer pair '. Means for amplifying genomic DNA is not limited to PCR, and it can be amplified by known amplification processes such as LAMP, LCR, NASBA and the like.

(Mutation detection)
The method for detecting the mutation of the obtained amplification product is not particularly limited, and various known methods are applied including the direct sequencing method. Examples of primers for direct sequencing include the primers of SEQ ID NOs: 4, 5, 6, 7 and 8. It is also possible to test by electrophoresis analysis of fragmented samples with restriction enzymes, FISH method for hybridizing DNA samples in solution, and the like. Other detection methods combining the Southern hybridization method, dot hybridization method (see J. Mol. Biol., 98 : 503-517, 1975, etc.), dideoxy base sequencing method, DNA amplification method [for example, PCR- Restriction fragment length polymorphism (RFLP), PCR-single-chain conformation polymorphism analysis (Proc. Natl. Acad. Sci., USA, 86 :
2766-2770, 1989, etc.), PCR-specific sequence oligonucleotide method (SSO), allele-specific oligonucleotide method using PCR-SSO and dot hybridization method (Nature, 324 : 163-166) , 1986 etc.)] and the like. Furthermore, it is also possible to detect simply by a nucleic acid chip or a nucleic acid array using an oligonucleotide probe.

Oligonucleotides of the primers used in the present invention can be easily synthesized using an automatic synthesizer such as a DNA synthesizer (Perkin Elmer) according to a conventional method. It can also be purified using a commercially available cartridge for purification.
Examples are given below to illustrate the present invention in more detail.

(Inspection target and sample preparation)
100 healthy Japanese subjects were examined. Genomic DNA was isolated and purified using Qiagen's Quiagen Blood DNA kit. For each, the exon 1 region and a part of the intron region of UGT1A4 were amplified by polymerase chain reaction (PCR) using the oligonucleotide primer pairs described in SEQ ID NOs: 2 and 3. The PCR was performed at 94 ° C for 5 minutes denaturation, 94 ° C for 1 minute, 62 ° C for 1 minute, 72 ° C for 2 minutes, 30 cycles. Thereafter, a final extension reaction was performed at 72 ° C. for 8 minutes. Amplified samples are subjected to sequencing using a dye terminator method using dichlororhodamine (dRhodamine Terminator Cycle
The sequence was confirmed by direct sequencing using Sequencing FS Ready Reaction Kit). As primers for sequence analysis, primers of SEQ ID NOs: 4, 5, 6, 7, and 8 were used.

(Allele analysis)
Alleles of the test target described in Example 1 were analyzed. The results are shown in FIG. The normal type was 68 cases (68%) among all 100 cases. In 32 cases (32%), 142T → G (L48V) mutation was observed, of which 142T → G (L48V) alone was found in only 2 cases, all of which were heterozygous mutations. Of the remaining 30 cases, in addition to 142T → G (L48V), both 448T → C (L150L) and 804G → A (P268P) were found to have duplicate mutations of 867 + 43C → T in the intron region. (Quadruple mutation). Among them, in one case, the duplication mutation was homozygous. The remaining 29 cases were all heterozygous, of which 2 were 804G → A (P268P) single mutation allele and the above-mentioned quadruple mutation allele. All of these mutations have not been reported so far.

From the human cDNA library, UGT1A4 cDNA was amplified by PCR using the primer pairs shown in SEQ ID NOs: 2 and 3, and incorporated into a pCR3.1 expression vector using a nucleated cell TA cloning kit (Invitrogen). Mutation introduction was performed using the Mutan Km kit (Takara). That is, the cDNA incorporated into the pCR3.1 expression vector was cleaved with two restriction enzymes Pst1 and BamH1 I and incorporated into the pkF18 vector (Takara). Mutations were introduced using the primer shown in SEQ ID NO: 9. The cDNA into which the mutation was introduced was excised from the pKF18 vector and ligated again to the pCR3.1 expression vector.
Each 3 μg of plasmid DNA was transfected into COS-7 cells using Gene PORTER transfection reagent (Gene Therapy System). After transfection of the transfected cells for 48 hours, the cells were collected and disrupted by sonication to prepare a cell homogenate. Cell homogenate UDP-glucuronyltransferase activity was measured using clozapine and UDP-glucuronic acid as substrates. Using UDP-glucuronic acid labeled with 14C, the product after reaction at 37 ° C. for 10 minutes was analyzed with TLC plastic sheet 5748 (Merck) to determine the enzyme activity. The results are shown in Table 1.

以上の結果、UGT1A４の酵素活性が１４２T→G（L48V）変異型では正常型の２０７．７％となり、本発明により、酵素活性の増大が予測できることが判った。

As a result, the enzyme activity of UGT1A4 was 207.7% of the normal type in the 142T → G (L48V) mutant, and it was found that the increase in enzyme activity can be predicted according to the present invention.

The present invention is used as a diagnostic kit used in the field of clinical tests necessary for diagnosis and treatment in addition to being used for prediction of drug efficacy and side effects in the development of new drugs.

Claims (6)

A method for testing a nucleic acid sequence of UDP-glucuronosyltransferase (UGT1A4), wherein the nucleic acid of UGT1A4 is characterized by examining a mutation from the 142nd t to g (L48V) of the sequence represented by SEQ ID NO: 1. Array inspection method. A method for examining or predicting the function and enzyme activity of a UGT1A4 enzyme molecule by examining the nucleic acid sequence of UGT1A4 according to claim 1. A method for examining drug metabolic capacity, jaundice, cancer, coronary artery disease or osteoporosis by the method according to claim 2.   The test method according to any one of claims 1 to 3, wherein any one or at least two of the oligonucleotide primers represented by SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 9 are used.   The inspection apparatus, reagent, or kit used for the inspection method of any one of Claims 1-5. A testing device, reagent or kit incorporating at least one oligonucleotide primer used in the testing method according to claim 4.