KR20040032838A - Genetic marker for decreased activity of cytochrome P450 3A7 protein - Google Patents

Abstract

PURPOSE: A genetic marker for decreased activity of cytochrome P450 3A7(CYP 3A7) protein and a method for detecting the same are provided, thereby anticipating drug response difference between individuals to a drug metabolized by CYP 2J2 through examination for possession of the genetic marker, and diagnosing a disease caused by the decreased activity of CYP 3A7. CONSTITUTION: A genetic marker for decreased activity of cytochrome P450 3A7 protein encodes a mutated CYP 3A7 protein in which a translation terminating codon(TAA) at amino acid position 55 in the amino acid sequence set forth in SEQ ID NO: 21 is formed, wherein a base T(Thymine) between base C(Cytosine) at nucleotide position 134 and base T(Thymine) at nucleotide position 135 in the nucleotide sequence set forth in SEQ ID NO: 20 is inserted. A method for detecting the genetic marker for decreased activity of cytochrome P450 3A7 protein comprises the steps of: (1) isolating DNA from a blood sample; (2) PCR amplifying the DNA with primers having the nucleotide sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 18; and (3) pyrosequencing the PCR product with a primer having the nucleotide sequence set forth in SEQ ID NO: 19 to confirm the mutation of the CYP 3A7 gene.

Description

Gene marker for reduced activity of cytochrome P450 3A7 protein

The present invention relates to a gene as a marker associated with a dysfunction of the CYP3A7 protein, and more particularly, to a CYP3A7 protein encoding a variant CYP3A7 protein which generates a translation termination codon (TAA) at the 55th amino acid position in a wild type CYP3A7 amino acid sequence. The present invention relates to genes as markers related to protein dysfunction.

Cytochrome P450 3A7 protein (CYP3A7), along with CYP3A4, CYP3A5 and CYP3A43, is a drug metabolic enzyme belonging to the CYP3 family. CYP3A7 is produced mainly in the liver of the fetus and accounts for about 30% of the total amount of cytochrome P450 enzymes. This enzyme is known to be expressed in the embryonic stage of early pregnancy. CYP3A7 enzyme is involved in the metabolism of foreign body foreign material coming from outside of the fetus. In addition, it is known to be involved in the internal metabolism of testosterone 6-hydroxylation and dehydroepiandristerone 3-sulfate 16-hydroxylation (Hakkola J et al., Harmacol Toxicol. 1998, 82 (5): 209-217).

Single nucleotide polymorphism (SNPs), on the other hand, is a phenomenon in which the bases that make up the genome are partially different from person to person, and human SNPs are 0.1% of the human genome of about 3 billion base pairs, or 1 per 1,000 bases. It is estimated that about 3 million dogs exist (Cooper, DN et al., Hum. Genet. 1985, 69: 201-205). SNPs are the most common form of DNA sequence variation, especially those that occur in the nucleotide sequence of the region where the gene is encoded (the region that makes the protein from the gene), which in humans is associated with many diseases, including cancer, hypertension, diabetes, and dementia. It is reported to be closely related (Sherrington et al. , Nature 1995, 375: 754-760; Kunz et al ., Hypertension. 1997, 30: 1331-1337; Rogaev et al ., Genomics 1997, 40: 415- 424). Thus, when comparing SNPs between the normal population and the patient population, if specific SNPs appear to be associated with the disease, these SNPs can be used as very useful genetic markers for specific diseases (Lai, E., Genomics 1998, 54: 31-38). That is, by identifying one or more sequences specific to the genetic disease in DNA extracted from the cells of the individual, it is possible to diagnose the onset and possibility of the genetic disease of the individual.

The gene of CYP3A7 is known to exhibit a genetic polymorphism. To date, four mutations have been found in the promoter portion of the gene (see www.imm.ki.se/cypalleles/cyp3a7.htm). However, there is no known single-base polymorphism at the splicing site involved in the exon or mRNA process of protein encoding. CYP3A7 is expressed not only in the fetus but also in some adults and is known to play a role in the metabolism of external drugs. Gene mutations that alter the function of this enzyme can have undesirable consequences on the metabolism of drugs or biomolecules. (Hakkola J et al., Pharmacol Toxicol. 1998 May; 82 (5): 209-17). Specifically, the CYP3A7 gene mutation may show differences in pharmacokinetics for some drugs, may be sensitive to external toxicants, and may alter normal production by altering the production of biomaterials required during development. Therefore, CYP3A7 gene mutations must be identified, and among them, the identification of functional gene mutations that can change the function of the enzyme is more important. In addition, in the case of many genes, the distribution of genetic polymorphisms is known to be different according to the species. Therefore, it is necessary to confirm the frequency of the CYP3A7 gene even if there are no intrinsic gene mutations frequently occurring in Koreans.

Therefore, the present inventors conducted a study for discovering a single base polymorphism affecting the activity of CYP3A7 from Korean genes. As a result, another base was inserted into the nucleotide sequence encoding the wild type CYP3A7 protein. The present invention has been accomplished by finding a single base mutation in which a translation termination codon is generated at an amino acid position, and finding that the single base mutation is a Korean specific DNA marker associated with the enzymatic abnormality of the CYP3A7 variant protein.

It is therefore an object of the present invention to provide a gene as a marker associated with dysfunction of the CYP3A7 protein, which encodes a variant CYP3A7 protein which produces a translational stop codon (TAA) at the 55th amino acid position in the wild type CYP3A7 amino acid sequence.

1 is a gel photograph showing the PCR amplification product of the CYP3A7 gene.

Lane 1: Size Marker Treated with Lambda DNA with Hind III

Lanes 2 to 5: P1 to P4

Figure 2 is a result of analyzing the heterozygous variant gene of CYP3A7 using an automatic sequence analyzer, the arrow indicates the position where thymine is inserted.

3 is a wild-type and heterozygous mutation using the pyroquencer (Pyrosequencer) whether the thymine (T) base is inserted between the 134th base (C) and 135th base (T) in the base sequence of SEQ ID NO: 20 The gene was confirmed.

In order to achieve the above object, the present invention is a labeling factor associated with the dysfunction of CYP3A7 protein, which encodes a variant CYP3A7 protein that generates a translation termination codon (TAA) at the 55th amino acid position in the amino acid sequence of SEQ ID NO: 21 Gene as.

Hereinafter, the present invention will be described in detail.

Genetic diseases develop due to changes in the nucleotide sequence of a normal gene. Such a change may be caused by substitution of one base of a gene with another base, or by insertion of another base between bases of the gene.

The present invention relates to a marker that can detect a genetic disease caused by insertion of another base between the bases of a gene. Specifically, the translation termination codon at the 55th amino acid position of the amino acid sequence of the wild type CYP3A7 protein of SEQ ID NO: 21. It is characterized by providing a single base mutation in which (TAA) is produced. Specifically, such a single base mutation may be caused by inserting a thymine (T) base between the 134th base (C) and the 135th base (T) in the nucleotide sequence of SEQ ID NO: 20 encoding the wild type CYP3A7 protein.

In one embodiment of the present invention, the entire nucleotide sequence of CYP3A7 genomic DNA (gDNA) represented by SEQ ID NO: 24 was analyzed using DNA isolated from the subject and an automatic sequencing analyzer (ABI 3700). A new single base polymorphism (SNP) was found.

Monobasic polymorphism according to the present invention is caused by the insertion of thymine (T) between the 22nd base (C) and 23rd base (T) of the second exon (7063 ~ 7155 bp) of CYP3A7 gDNA of SEQ ID NO: 24 When this corresponds to the CYP3A7 cDNA nucleotide sequence represented by SEQ ID NO: 20, it can be seen that the thymine (T) base is inserted between the 134th base (C) and the 135th base (T) (see FIG. 2). ).

When thymine is inserted in positions 134 and 135 of the CYP3A7 cDNA nucleotide sequence represented by SEQ ID NO: 20, the nucleotide sequence shown in SEQ ID NO: 22 will be obtained, and as shown in SEQ ID NO: 23, thereafter, A translational stop codon of TAA is generated at the 55th amino acid position that emerges. Therefore, the gene containing the mutation does not produce a complete form of the CYP3A7 enzyme but only produces a protein fragment that lacks the function. In a subject with the genetic mutation, the enzyme activity of the CYP3A7 is deficient, resulting in metabolic abnormalities of drugs or biological molecules. May result.

Therefore, by examining the possession of the CYP3A7 mutant gene, it is possible not only to predict individual drug response differences of drugs metabolized by CYP3A7, but also to diagnose diseases related to CYP3A7 protein abnormalities.

As a method for testing a gene as a marker associated with a dysfunction of the CYP3A7 protein according to the present invention, all conventionally known SNPs test methods can be used, and specifically, polymerase chain reaction-restriction fragment length formation (Polymerase Chain Reaction). Restriction Fragment Length Polymorphism (PCR-RFLP), Single Strand Conformation Polymorphism (SSCP), DNA sequencing, pyrosequencing, denaturation gradient gel electrophoresis gel electrophoresis), DNA chips using oligonucleotides, and the like can be used.

Specifically, pyro sequencing is a method of detecting which base is newly used according to each genotype during the DNA polymerization reaction.

In the case of bases used for DNA polymerization, pyrophosphate is advantageous, which is converted into ATP in the presence of adenosine 5'-phosphosulfate and luminescence reaction by luciferase activated by ATP. This is measured. Since the height of each peak is proportional to the number of bases added, the homozygote is twice the heterozygote peak. If the added base is not used for DNA synthesis, each genotype can be known because pyrophosphate is not advantageous and does not cause a luminescence reaction.

Specifically, the method for detecting a gene as a marker associated with dysfunction of the CYP2J2 protein of the present invention comprises the steps of: (1) separating DNA from a blood sample of a subject; (2) amplifying the separated DNA by performing PCR using primers of SEQ ID NO: 17 and SEQ ID NO: 18; And (3) identifying the mutation of the CYP3A7 gene by pyrosequencing using the primer of SEQ ID NO: 19 as a template of the PCR product of step (2).

In another embodiment of the present invention, the CYP3A7 gene mutation was searched for in the subject using the detection method. As a result, one of the two strands of DNA has a variant and one strand has a wild type. It could be confirmed (see Fig. 3).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Amplification of the CYP3A7 Gene

After separating blood from 48 healthy subjects, DNA was isolated using Qiagen's Genomic DNA Separation Kit according to the instructions.

The entire nucleotide sequence of CYP3A7 gDNA represented by SEQ ID NO: 24 and the gDNA sequence isolated from the subject were analyzed using an automatic sequencing analyzer (ABI 3700) to find a new single base polymorphism (SNP). To determine whether this nucleotide polymorphism is located on the strand of the CYP3A7 gene, haplotype determination, whether there are no other gene mutations on the same DNA strand, or whether it originates from pseudogenes located on different parts of the chromosome. One strand of DNA from the subject's DNA containing the mutation was cloned in full size using a PCR reaction and then sequenced.

As listed in SEQ ID NO: 24, CYP3A7 gDNA has 13 exons and has very long introns between each exon, resulting in 33,226 bp of total gene length, resulting in four CYP3A7 gDNAs (3A7-P1, PCR was performed by dividing into fragments of P2, P3, P4). The primer pair used for each PCR, the binding position of the primer pair, and the size of the amplified product are shown in Table 1 below. In Table 1 below, “IVS” means an intron (intervening sequence), a number after IVS is a number indicating the number of introns, and also a “+ number” following an intron number (eg IVS2, IVS8, etc.). Indicates the relative position in the dowstream direction when the start of the intron is +1, and the “-number” following the intron number is upstream when the last position of the intron is -1. Relative position in the () direction.

CYP3A7 gene fragment Primer pairs Position Size (bp) 3A7-P1 SEQ ID NO: 1 -665 to -646 4978 SEQ ID NO: 2 IVS2 + 156 to IVS2 + 175 3A7-P2 SEQ ID NO: 3 IVS2-135-IVS2-112 8217 SEQ ID NO: 4 IVS8 + 269-IVS8 + 288 3A7-P3 SEQ ID NO: 5 IVS8-254 ~ IVS8-235 4843 SEQ ID NO: 6 IVS11 + 63 to IVS11 + 82 3A7-P4 SEQ ID NO: 7 IVS10-231 to IVS10-211 5596 SEQ ID NO: 8 IVS13 + 1128 to IVS13 + 1147

On the other hand, PCR reaction conditions of each CYP3A7 gene fragment are shown in Table 2 below.

PCR products Reaction condition 3A7-P1 94 ° C 1 minute, (98 ° C 10 seconds, 58 ° C 30 seconds, 72 ° C 5 minutes) 30cycles, 72 ° C 10 minutes 3A7-P2 94 ° C 1 minute, (98 ° C 10 seconds, 58 ° C 30 seconds, 72 ° C 8 minutes) 30cycles, 72 ° C 10 minutes 3A7-P3 94 ° C 1 minute, (98 ° C 10 seconds, 60 ° C 30 seconds, 72 ° C 5 minutes) 30cycles, 72 ° C 10 minutes 3A7-P4 94 ° C 1 minute, (98 ° C 10 seconds, 56 ° C 30 seconds, 72 ° C 5 minutes) 30cycles, 72 ° C 10 minutes

The PCR product after the PCR reaction was confirmed by gel electrophoresis. As a result, as shown in Figure 1, it was confirmed that all the PCR products of the 3A7-P1 to 3A7-P4 amplified.

Example 2 CYP3A7 Sequencing

Each PCR product obtained in Example 1 was confirmed by an automatic base sequence analyzer (ABI 3700). The sequencing primer sequence used at this time is shown in Table 3 below.

PCR products Sequencing primers 3A7-P1 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 3A7-P2 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 3A7-P3 SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14 3A7-P4 SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 15

Autobase sequencing revealed no other mutations in the CYP3A7 gene DNA strand. However, as shown in FIG. 2, as in the first discovery, the mutant gene was confirmed that thymine (T) was inserted between the 22nd base and the 23rd base of the second exon. This corresponds to between 7084 and 7085 bases in the total CYP3A7 gDNA represented by SEQ ID NO: 24, and between 134 and 135 bases in the CYP3A7 cDNA represented by SEQ ID NO: 20.

On the other hand, a subject having such a mutant gene, as shown in Figure 3, one of the two strands of DNA was found to have a variant, one strand has a wild type.

When the thymine (T) base is inserted between the 134th base (C) and the 135th base (T) in the nucleotide sequence of SEQ ID NO: 20, the translation termination codon of TAA is generated at the 55th amino acid position subsequent thereto (sequence) Number 23). Thus, the gene containing the mutation does not produce a complete form of the CYP3A7 enzyme, but only produces a protein fragment lacking function. In subjects with such genetic mutations, the enzyme activity of CYP3A7 is deficient and may cause metabolic abnormalities of drugs or biological molecules.

Example 3 Detection of Mutants Using PCR / Pyro Sequencing

We searched for CYP3A7 gene mutations in blood samples of subjects. After preparing a primer pair for PCR so that a PCR product containing a second exon of CYP3A7 gDNA containing a mutation was produced, PCR was performed. As primer pairs, primers of SEQ ID NO: 17 and SEQ ID NO: 18 were used, and the 5 'side of SEQ ID NO: 17 was labeled with biotin. The PCR reaction was denatured at 94 ° C. for 5 minutes, and then the reaction was repeated 35 times for 30 seconds at 94 ° C., 30 seconds at 60 ° C., and 30 seconds at 72 ° C., and finally amplified at 72 ° C. for 5 minutes.

Then, it was confirmed that only one type of PCR product was generated by electrophoresis. The PCR product was used as a template to determine the presence or absence of mutations in a pyrosequencer (Pyrosequencing) using a sequencing primer of SEQ ID NO: 19. It was.

As a result, as shown in Figure 3, it can be seen that the two genotypes are distinct. In addition, pyro sequencing method was confirmed that the presence or absence of genetic mutation within 40 minutes starting from the PCR product.

Mutant genes identified through sequencing were used as controls to search for the presence of mutant genes in Koreans, Vietnamese, African Americans, and Westerners. The DNA of other races other than Koreans was provided by the Gene Bank, which is being built at the Inje University School of Medicine.

A total of 544 persons, 185 Koreans, 159 Vietnamese, 100 African-Americans and 100 Westerners, were tested for the presence of the mutant gene using a pyrosequencer. No subjects with the mutant gene were found. In conclusion, the frequency of this new gene mutation was 0.21% in Koreans.

As described above, the monobasic polymorphism found in the gene of cytochrome P450 3A7 (CYP3A7) first discovered in Korean can be applied to predict individual drug response differences of drugs metabolized by CYP3A7, and furthermore, CYP3A7 protein aberration-related diseases. It can be applied to diagnose.

Claims (3)

A gene as a marker associated with dysfunction of the CYP3A7 protein, which encodes a variant CYP3A7 protein which generates a translation termination codon (TAA) at the 55th amino acid position in the amino acid sequence of SEQ ID NO: 21. The gene according to claim 1, wherein a thymine (T) base is inserted between a 134th base (C) and a 135th base (T) in the nucleotide sequence of SEQ ID NO: 20. A method for detecting a gene as a marker associated with dysfunction of the CYP3A7 protein comprising the following steps: (1) isolating DNA from a blood sample of the subject; (2) amplifying the separated DNA by performing PCR using primers of SEQ ID NO: 17 and SEQ ID NO: 18; And (3) confirming the presence or absence of mutation of the CYP3A7 gene by pyrosequencing method using the primer of SEQ ID NO: 19 as a template of the PCR product of step (2).