WO2007085127A1

WO2007085127A1 - A method for prognosing therapeutic effectiveness of acute angina pectoris with nitroglycerin and a kit therefor

Info

Publication number: WO2007085127A1
Application number: PCT/CN2006/000188
Authority: WO
Inventors: Li Jin; Wei Huang; Yifeng Li
Original assignee: National Human Genome Center At Shanghai; Fudan University
Priority date: 2006-01-27
Filing date: 2006-01-27
Publication date: 2007-08-02

Abstract

A method for prognosing therapeutic effectiveness of acute angina pectoris with nitroglycerin comprises detecting whether a variation occurs in acetaldehyde dehydrogenase 2 (ALDH2) gene, its transcript and/or encoded protein of an individual as compared with that of normal control. The presence of variation demonstrates that for this individual, the therapeutic effectiveness of acute angina pectoris with nitroglycerin is lower than that of ordinary patient with acute angina pectoris. There is also a kit for such method.

Description

Method and kit for detecting glycerol in treating acute angina pectoris

Technical field

The present invention relates to the fields of molecular biology and medicine. More specifically, it relates to single nucleotide polymorphism (SNP) of acetaldehyde dehydrogenase 2 (ALDH2) and its correlation with the efficacy of nitroglycerin in the treatment of acute angina pectoris. The invention also relates to methods and kits for detecting these SNPs. Background technique

Since the first clinical application in 1876, Nitroglycerin (NTG) has been widely used to treat acute angina. Sublingual gland administration of nitroglycerin has become the standard treatment for acute angina. In general, sublingual gland administration of nitroglycerin can alleviate the symptoms of acute angina within 5 to 10 minutes. The principle of physiological treatment of nitroglycerin is mainly explained by its ability to release pharmacologically active nitrogen oxides (NO or NO congener), thereby activating and promoting the downstream cyclic guanosine monophosphate (cGMP) pathway to relieve vascular smooth muscle. .

Acute angina is an extremely dangerous disease that can be life-threatening if not treated in time. Although nitroglycerin has been widely used to treat acute angina pectoris, it is still clinically found that some patients with acute angina have no significant effect on nitroglycerin administration in the sublingual gland. This suggests that these people may be insensitive to nitroglycerin therapy for a variety of reasons.

Attempts have been made to address a variety of causes that may lead to failure of nitroglycerin therapy, including environmental differences, individual differences, and eating habits.

The acetaldehyde dehydrogenase 2 gene is a member of a family of NAD (P)-dependent aldehyde dehydrogenase genes. The main role of this gene family is to oxidize aldehydes in physiological environments. In addition to the activity of dehydrogenase, the acetaldehyde dehydrogenase 2 gene has a relatively strong esterase activity. It promotes the hydrolysis of nitroglycerin to 1,2-dinitroglycerol (1,2-GDN) and nitrite ions ( ₂ ).

In 2002, Chen. ZQ et al. found that the vascular expansion of cGMP-dependent nitroglycerin could be blocked by inhibitors of ALDH2 protein, both in vivo and in vitro. This suggests that the acetaldehyde dehydrogenase 2 gene is involved in the physiological role of cGMP-dependent nitroglycerin in expanding blood vessels and plays an important role.

However, to date, the real cause of nitroglycerin treatment failure has not been confirmed. In summary, people also understand the reasons for the effectiveness of nitroglycerin in the treatment of acute angina pectoris Very little. In order to specifically use nitroglycerin, there is an urgent need in the art to develop methods and kits for predicting the effectiveness of nitroglycerin in the treatment of acute angina. Summary of the invention

It is an object of the present invention to provide a method and kit for predetermining the effectiveness of nitroglycerin in the treatment of acute angina pectoris. In a first aspect of the invention, there is provided a method of detecting in vitro whether a sample has a single nucleotide polymorphism of the acetate dehydrogenase 2 gene ALDH2, comprising the steps of:

(a) amplifying a sample of the ALDH2 gene with an ALDH2 gene-specific primer to obtain an amplification product;

(b) Detection of the presence of the following single nucleotide polymorphisms in the amplified product:

1951 G→A;

Wherein, the nucleotide position number is based on SEQ ID Ν0: 1 ο

In another preferred embodiment, the gene-specific primer has the sequences of SEQ ID NO: 3 and 4. In another preferred embodiment, the amplification product is 50-3000 bp in length and contains position 1951 in SEQ ID NO: 1.

In a second aspect of the invention, there is provided a kit for predicting the effectiveness of nitroglycerin in the treatment of acute angina pectoris comprising a primer which specifically amplifies an ALDH2 gene or transcript, said primer amplifying a length of 50. - 3000 bp, and contains the amplification product at position 1951 of SEQ ID NO: 1.

In another preferred embodiment, the kit further comprises a reagent selected from the group consisting of -

(a) a probe that binds to a mutation at position 1951 of SEQ ID NO: 1;

(b) A mutant restriction endonuclease at position 1951 in SEQ ID NO: 1.

In another preferred embodiment, the mutation is a single nucleotide polymorphism as follows:

1951 G→A;

Wherein, the nucleotide position number is based on SEQ ID NO: 1.

In another preferred embodiment, the primer has the sequence of SEQ ID NO: 3 and 4.

In a third aspect of the invention, there is provided a method of predicting the effectiveness of nitroglycerin in the treatment of acute angina pectoris comprising the steps of: detecting an ALDH2 gene, a transcript and/or a protein of a subject to be examined, and interacting with normal ALDH2 Comparison of genes, transcripts and/or proteins,

The difference indicates that nitroglycerin is less effective for treating acute angina in this individual than in the general acute angina population. In another preferred embodiment, the gene or transcript of ALDH2 is detected and compared to the normal ALDH2 nucleotide sequence.

In another preferred embodiment, the difference is the following single nucleotide polymorphism -

1951 G-A;

Wherein, the nucleotide position number is based on SEQ ID Ν0: 1 ο

In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising 0. 01-9 1:% of 1,2-dinitroglycerol (1,2-GDN) and nitrite ion (N(V) And a pharmaceutically acceptable carrier. Alternatively, the composition comprises (a) a safe and effective amount (0. 01-99 wt%) of nitroglycerin, (b) an effective amount of hydrolyzing nitroglycerin to 1,2-dinitrate Wild type acetaldehyde dehydrogenase 2 of glycerol (1,2-GDN) and nitrite ion (N0 ₂ - ) (generally, the molar ratio of acetaldehyde dehydrogenase 2 to nitroglycerin is 1: 10, 000 to 1 : 1, 000, 000), and a pharmaceutically acceptable carrier.

The inventors have conducted intensive and extensive research to measure and analyze a large number of candidate gene SNPs. It was first discovered and proved that some SNPs of ALDH2 gene are closely related to the efficacy of nitroglycerin in the treatment of acute angina pectoris. The change of ALDH2 will lead to a decrease in the efficacy of nitroglycerin in the treatment of acute angina pectoris. The correlation study shows that the SNP at 1951 of ALM2 (1951) There is a significant difference in the distribution of G→A) between the case and the control group (0.01), so it can be used as a specific SNP for predicting the effectiveness of nitroglycerin in the treatment of acute angina. The present invention has been completed on this basis.

ALDH2 gene

The detailed sequence of the acetaldehyde dehydrogenase 2 gene (ALDH2) can be found in the nucleotide sequence of accession number NT-009775 (see URL

Http: 〃 www. ncbi. nlm. nih. gov/) , as shown in SEQ ID NO: 1. The encoded amino acid sequence is shown in SEQ ID NO: 2.

The acetaldehyde dehydrogenase 2 gene is a member of a family of NAD (P)-dependent aldehyde dehydrogenase genes. The main role of this gene family is to oxidize aldehydes in physiological environments. In addition to the activity of dehydrogenase, the acetaldehyde dehydrogenase 2 gene has a relatively strong esterase activity. It promotes the hydrolysis of nitroglycerin to 1,2-dinitroglycerol (1,2-GDN) and nitrite ions (N0 ₂ -).

Studies of the present invention have shown that when acetaldehyde dehydrogenase 2 is mutated to cause a decrease in activity, the expansion of vascular nitrate may be affected or even blocked, resulting in a decrease in the efficacy of nitroglycerin.

As used herein, "ALDH2 gene" refers to a polynucleotide molecule encoding acetaldehyde dehydrogenase 2, including DNA and RNA.

As used herein, "ALDH2 protein" refers to acetaldehyde dehydrogenase 2 protein.

As used herein, the aldehyde dehydrogenase 2 gene is divided into two types of alleles, namely aldehyde dehydrogenase 2 gene type 1 (ALDH2*1) and polymorphisms with a polymorphic site G. Point A is acetaldehyde dehydrogenase 2 gene type 2 (ALDH2*2). The present inventors measured and analyzed a large number of candidate gene SNPs, in which almost the entire region of the ALDH2 gene was sequenced, and many SNPs were found, most of which were not related to the effectiveness of nitroglycerin in the treatment of acute angina pectoris. However, association studies have shown that 1951 G-A is a very high SNP associated with the efficacy of nitroglycerin in the treatment of acute angina. The SNP changed the reading frame from GAA to AAA, which caused a change in 504 amino acids from Glu to Lys, which in turn led to the activity of ALDH2, and ultimately led to the carrier's nitroglycerin treatment of acute angina was significantly lower than ordinary Acute angina population.

Based on the novel findings of the present invention, ALDH2 proteins or polypeptides have many new uses. These uses include, but are not limited to, adjuvant therapy for acute angina, such as in combination with nitroglycerin for the treatment of acute angina.

In another aspect, the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, which are specific for human ALDH2 DNA or a polypeptide encoded by the fragment thereof. Here, "specificity" means that an antibody binds to a human ALDH2 gene product or fragment. Preferably, those antibodies which bind to a human ALDH2 gene product or fragment but which do not recognize and bind to other non-related antigen molecules. The antibodies of the present invention include those capable of binding to and inhibiting the human ALDH2 protein, as well as those which do not affect the function of the human ALDH2 protein.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or chimeric antibodies. ■

Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified human ALDH2 gene product or a fragment thereof having antigenicity can be administered to an animal to induce production of a polyclonal antibody. Similarly, cells expressing human ALDH2 protein or its antigenic fragment can be used to immunize animals to produce antibodies. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.

The antibody of the invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology. Antibodies of the invention include antibodies that block the function of human ALDH2 protein and do not affect human ALDH2 Protein-functional antibodies. The various antibodies of the invention can be obtained by conventional immunological techniques using fragments or functional regions of the human ALDH2 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. An antibody that binds to an unmodified form of the human ALDH2 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (eg, E. Col i); an antibody that binds to a post-translationally modified form (eg, glycosylation or phosphorylation) The protein or polypeptide) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell such as yeast or insect cells.

Antibodies against human ALDH2 protein can be used in immunohistochemistry to detect the number and/or mutation of human ALDH2 protein in biopsy specimens. A preferred anti-ALDH2 antibody is an antibody that does not recognize normal ALDH2 but recognizes the mutant ALDH2, or an antibody that recognizes normal ALDH2 (i.e., wild type) but does not recognize mutant ALDH2. Using these antibodies, it is convenient to predict the effectiveness of nitroglycerin in the treatment of acute angina at the protein level.

Using the ALDH2 protein of the present invention, substances which interact with the ALDH2 protein, such as inhibitors, agonists or antagonists, can be screened by various conventional screening methods.

The ALDH2 protein of the present invention, an agonist thereof and the like, can provide different effects when administered (administered) therapeutically. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, sublingual, intramuscular, intravenous, or subcutaneous administration.

Normal ALDH2 can be used directly for the treatment of diseases, for example, in the treatment of acute angina pectoris with nitroglycerin. In the case of using the ALDH2 protein inhibitor of the present invention, other agents for treating the efficacy of nitroglycerin for the treatment of acute angina can also be used simultaneously.

The present invention also provides a pharmaceutical composition comprising a safe and effective amount of the ALDH2 protein of the present invention and nitroglycerin, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, ethanol, and combinations thereof. The pharmaceutical preparation should be matched to the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as solutions, tablets and capsules are preferably prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 0.1 microgram per kilogram of body weight per day to about 10 milligrams per kilogram of body weight.

When a pharmaceutical composition is used, a safe and effective amount of the ALDH2 protein or an agonist thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 0.1 μg/kg body weight, and in most cases Not more than about 10 mg/kg body weight, preferably the dose is about 0.1 μg/kg body weight to about 100 μg/kg body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The invention also relates to a test method for quantifying and localizing the detection of human ALDH2 protein levels. These assays are well known in the art and include ELISA and the like.

A method for detecting the presence or absence of ALDH2 protein in a sample is to detect the specific antibody of the ALDH2 protein, which comprises: contacting the sample with an ALDH2 protein-specific antibody; observing whether an antibody complex is formed, and forming an antibody complex means a sample There is an ALDH2 protein present.

Polynucleotides of the ALDH2 protein can be used to predetermine the effectiveness of nitroglycerin in the treatment of acute angina. For example, the ALDH2 DNA sequence can be used to hybridize to a biopsy specimen to determine abnormal expression of the ALDH2 protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and related kits are commercially available. A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as "gene chip") for analyzing differential expression analysis and gene detection of genes in tissues. The ALDH2 protein transcriptional product can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using ALDH2 protein-specific primers.

Detection of mutations in the ALDH2 gene can also be used to predetermine the efficacy of nitroglycerin in the treatment of acute angina. Detection can be directed to cDNA as well as to genomic DNA. The ALDH2 protein mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type ALDH2 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of the protein, so Northern blotting and Western blotting can be used to indirectly determine whether the gene has a mutation.

The most convenient method for detecting the SNP of the present invention is to obtain an amplification product by amplifying a sample ALDH2 gene with an ALDH2 gene-specific primer; and then detecting whether the following single nucleotide polymorphism exists in the amplification product: 1951 G →A, wherein the nucleotide position number is based on SEQ ID NO: 1.

It should be understood that, after the present invention first reveals the correlation between the SNP of the ALDH2 gene and the therapeutic effect of nitroglycerin in the treatment of acute angina pectoris, those skilled in the art can conveniently design an amplification product that specifically amplifies the position containing the SNP. Then, whether or not there is 1951 G-A is determined by sequencing or the like. Typically, the primers are 15 to 50 bp in length, preferably 20 to 30 bp. Although it is preferred that the primer is fully complementary to the template sequence, it will be appreciated by those skilled in the art that in the presence of a certain non-complementary primer (especially the 5' end of the primer), the primer can also be specifically amplified (ie only Amplify the desired fragment). Kits containing these primers and methods of using the same are within the scope of the present invention as long as the primers are amplified The amplified product contains the corresponding position of the SNP of the present invention. A preferred primer pair has the sequence of SEQ ID NO: 3 and 4.

Although the length of the amplification product is not particularly limited, the length of the amplification product is usually 50 - 3000 bp, preferably 150 - 2000 bp, more preferably 200 - 1000 bp. These amplification products should all contain the 1951th position in SEQ ID NO: 1. Since the SNP of the present invention has a very high correlation with the efficacy of nitroglycerin in the treatment of acute angina pectoris, it can be used not only for early and more accurate pre-determination of the effectiveness of nitroglycerin in the treatment of acute angina, but also to enable the carrier to be proactively Take reasonable precautions before or at the time of onset (eg use other effective drugs such as 1,2-dinitroglycerol (1,2-GDN) and nitrite (N(V), or use nitroglycerin) At the same time, the use of acetaldehyde dehydrogenase 2), thereby significantly improving the therapeutic effect on patients with acute angina pectoris, and therefore has extremely significant application value and social benefits. The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments It is intended to illustrate the invention and is not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor) The conditions described in the Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer.

1. 1 research object

Eighty Chinese Hans were selected and patients with coronary heart disease were diagnosed. They all used nitroglycerin as an angina pectoris. Based on the effect of sublingual nitroglycerin administration, these patients can be divided into effective groups that are effective within ten minutes, and ineffective groups that are not effective within ten minutes. The effective group consisted of 59 patients, including 47 males and 12 females. There were 21 patients in the ineffective group, including 13 males and 8 females. In these patients, the effect of drug administration was not significantly related to the gender of the patient (p=0.107).

1. 2 Experimental methods and results

1. 2. 1 DNA extraction

DNA was extracted from human blood by conventional phenol chloroform method and adjusted to 20 _ng / ul for routine PCR amplification. 1. 2. 2 PCR and sequencing primer design

The following primers were designed and synthesized based on the sequence of the ALDH2 gene in GenBank. Specific primers are shown below.

Table 1 Primer Sequence Listing

1. 2. 3 PCR amplification of ALDH2 gene

Using the extracted DNA as a template, the Taq enzyme was used for PCR amplification on a GeneAmp 9700 PCR machine using the Touchdown program. The reaction conditions were: 94 Ό pre-denaturation for 2 minutes, 94 Ό denaturation for 30 seconds, 63 Ό annealing for 40 seconds, and 72 ° C for 40 seconds for a total of 10 cycles, each cycle annealing temperature was decreased by 0.5 ° C; after 94 Ό denaturation for 30 seconds, 58 Ό annealing for 40 seconds, 72 ° C for 40 seconds, a total of 30 cycles; the last 72 Ό extension for 7 minutes. The PCR amplification products were verified by agarose gel electrophoresis.

As a result, an amplification product of 358 bp was obtained.

1. 2. 4 SNP discovery and detection,

The PCR product was purified by Resin resin and subjected to bidirectional sequencing by fluorescent label end termination using ABI-PRISMTM 377 DNA Sequencer (Applied Biosystems (ABI), USA), using Polyphred software (University of Washington, USA http://ddog. Mbt. Washington, edu/

Polyphred. html) performs sequence interpretation and SNP confirmation.

As a result, it was found that the following SNP was present: 1951 G→A.

1. 2. 5 SNP genotyping and analysis

These 80 patients were genotyped against the 1951 G/A site in the acetaldehyde dehydrogenase 2 gene using the results of PCR and sequencing as above.

The results showed that in the effective group, the number of homozygous aldehyde dehydrogenase 2 gene type 1 (ALDH2*1) individuals (ie, G/G at 1951) was 40; in the ineffective group, acetaldehyde dehydrogenase The number of homozygous individuals of the 2 gene type 1 (ALDH2*1) was 7. The number of homozygous aldehyde dehydrogenase (ALDH2 * 1) type 12 gene in two different groups, with significant difference (χ2 = 7. 59, ρ = 0. 006) 0 results demonstrate that the tongue The effect of nitroglycerin administration on acute angina pectoris, the individual homozygous for aldehyde dehydrogenase 2 gene type 1 (ALDH2*1) An individual that is homozygous for aldehyde dehydrogenase 2 gene type 2 (ALDH2*2) (ie, A1 at 1951) or heterozygous for type 1 and type 2 of aldehyde dehydrogenase 2 gene (ie, 1951) Bits are G/A) to be more effective, and there is a significant difference between the two. Example 2

Activity determination of wild type and mutant acetaldehyde dehydrogenase 2

From the individuals heterozygous for acetaldehyde dehydrogenase 2 genotype, reverse transcription (DNA) of two different genotypes of wild-type and 1951 G-A mutants were obtained by reverse transcription and sequenced. The resulting sequence was verified. Subsequently, nested PCR (nest-PCR) was used to amplify the target sequence and introduced a restriction site. The amplified sequence was then recombined into a plasmid (recombinant plasraid) and the plasmid was transfected into insect cell sf (purchased from Invitrogen). The sequence of the recombinant plasmid was also verified by sequencing, SEQ ID NO: 5 (wild type, position 1510 is G) and SEQ ID NO: 6 (mutant type, position 1510 is A). The expression of the acetaldehyde dehydrogenase 2 gene of the plasmid infected into the cells was verified by conventional immunoblotting.

Recombinantly expressed acetaldehyde dehydrogenase 2 gene protein, purified by affinity chromatography, can be stained with 12% acrylamide gel and stained with Coomassie blue to see an egg of approximately 54kDa.

3

White. 〇

曰 ω

Recombinantly expressed two different genotypes of acetaldehyde dehydrogenase 2 gene protein, and directly from acetaldehyde dehydrogenase 2 gene type 1 homozygous individual and acetaldehyde dehydrogenase 2 gene type 1 and type 2 heterozygous The purified acetaldehyde dehydrogenase 2 gene protein obtained by the individual was tested for the ability of these four samples to convert nitroglycerin by radiochemical methods.

The results are as follows:

Kinetic properties of two allelic types of proteins _: enzyme genotype dehydrogenase activity transforms nitroglycerin activity

(U/mg) Km

(μΜ)

G/G 0. 586 8. 20 5. 14

G/A 0. 075 20. 24 1. 12

G (transfected) 0. 544 11. 32 4. 18

A (transfected 1951G/A) 0. 012 25. 45 1. 08 It was found that the protein of type 1 (ie, wild type) of acetaldehyde dehydrogenase 2 gene was significantly lower than the protein of type 2 of acetaldehyde dehydrogenase 2 gene, with a lower Km and a higher Vmax in the conversion of nitroglycerin. . The protein homozygous for the acetaldehyde dehydrogenase 2 gene type 1 protein also has a lower Km and a higher Vmax than the type 1 and type 2 heterozygous protein.

Thus, the protein of acetaldehyde dehydrogenase 2 gene type 1 has a higher physiological activity for converting nitroglycerin. The acetaldehyde dehydrogenase 2 gene type 2 protein, due to the mutation of 1951G-A, changes the amino acid sequence of the protein, affecting the function and activity of the protein. The protein of type 2 of acetaldehyde dehydrogenase 2 gene can not effectively transform nitroglycerin, which affects the effect of nitroglycerin on the downstream cGMP pathway, and can not effectively relax vascular smooth muscle, thus reducing the effect of nitroglycerin on acute angina pectoris. . Example 3

Kit for predicting the effectiveness of nitroglycerin in the treatment of acute angina pectoris

As described in Example 1, the mutation of 1951 G-A in SEQ ID NO: 1 is closely related to the efficacy of nitroglycerin in the treatment of acute angina pectoris. Therefore, based on this mutation, the ALDH2 gene-specific primer can be designed to be predicted by amplification using the DNA of the individual to be tested as a template.

Prepare a kit (100 person times) containing:

3 ml of blood of the male patient to be tested is taken, and DNA is extracted from the blood using a conventional method (or using a specific kit). The PCR primers in the predictive kit for the therapeutic effect of nitroglycerin for acute angina pectoris were diluted to limol/il, and the extracted DNA was used as a template to carry out a PCR reaction with the provided primers. After purification of the PCR product, the ABI-PRISMTM 377 DNA sequencer was used for bidirectional sequencing of the fluorescently labeled end-stopping method, and sequence interpretation and SNP confirmation were performed using Polyphred software.

Alternatively, chromatographic analysis of the amplified product with a normal control by denaturing high performance liquid chromatography (DHPLC) can also detect 1951 G→A.

The results showed that the efficacy of nitroglycerin in the treatment of acute angina pectoris (with a ten-minute effective rate) of the subjects with 1951 G-A was lower than that of the general acute angina pectoris. Example 4

Correlation of other SNPs of acetaldehyde dehydrogenase 2 with the efficacy of nitroglycerin in the treatment of acute angina pectoris In addition to the 1951 G-A, the following 9 SNPs in the ALDH2 gene were detected by the method described in Example 1:

These SNPs were subjected to SNP genotyping and analysis in the same manner as in Example 1. The results showed that there was no statistically significant correlation between the efficacy of these SNPs and nitroglycerin in the treatment of acute angina. discuss

In exon 12 of the aldehyde dehydrogenase 2 gene (ALDH2), there is a single nucleotide polymorphism site (SNP), which is the 1951 guanosine monophosphate in SEQ ID NO: 1. Polymorphism of adenylate (G/A). This SNP site changes the amino acid 504 of the acetaldehyde dehydrogenase 2 protein from glutamic acid (Glu) to lysine (Lys) (see SEQ ID NO: 2). From this SNP, the aldehyde dehydrogenase 2 gene is divided into two types of alleles, namely aldehyde dehydrogenase 2 gene type 1 (ALDH2*1) and polymorphisms with a polymorphic site of G. The point is A aldehyde dehydrogenase 2 gene type 2 (ALDH2*2).

This SNP has a relatively high frequency in the Asian population, including the Chinese, about 30% left. Right. Since it changes the amino acid sequence of the acetaldehyde dehydrogenase 2 gene protein, this SNP has a functional role. The acetaldehyde dehydrogenase 2 gene type 1 has normal enzymatic activity, while the acetaldehyde dehydrogenase 2 gene type 2 produces an enzyme with active defects. The normally active enzyme produced by acetaldehyde dehydrogenase 2 gene type 1 (ALDH2*1) can promote the downstream reaction and cause nitroglycerin to function as a relaxing blood vessel. The enzyme with no normal activity generated by aldehyde dehydrogenase 2 gene type 2 (ALDH2*2) can not normally promote the downstream reaction, so that the vasodilating action of nitroglycerin is weakened or even disappeared.

The present invention suggests that acetaldehyde dehydrogenase 2 gene type 1 can be administered in combination with nitroglycerin, or in patients with acute angina having 1951 G-A mutation, 1,2-dinitro can be used in addition to nitroglycerin. Drugs such as glycerol (1, 2-GDN) and nitrite ion (N0 ₂ - ). All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

A method for detecting, in vitro, a sample for the presence of a single nucleotide polymorphism of the aldehyde dehydrogenase 2 gene ALDH2, comprising the steps of:

1951 G→A;

Wherein, the nucleotide position number is based on SEQ ID NO: 1.

2. The method of claim 1, wherein said gene-specific primer has the sequence of SEQ ID NO: 3 and 4.

The method according to claim 1, wherein the amplification product has a length of 50 to 3000 bp and contains the 1951th position in SEQ ID NO: 1.

A kit for predicting the effectiveness of nitroglycerin for treating acute angina pectoris, comprising: a primer for specifically amplifying an ALDH2 gene or a transcript, said primer having a length of 50-3000 bp and comprising Amplification product at position 1951 in SEQ ID NO: 1.

The kit according to claim 4, further comprising a reagent selected from the group consisting of:

(a) a probe that binds to a mutation at position 1951 of SEQ ID NO: 1;

(b) A mutant restriction endonuclease at position 1951 in SEQ ID NO: 1.

The kit according to claim 5, wherein the mutation is the following single nucleotide polymorphism -

1951 G→A;

Wherein, the nucleotide position number is based on SEQ ID NO: 1.

The kit according to claim 4, wherein the primer has the sequences of SEQ ID NOs -.3 and 4.

8. A method of predicting the effectiveness of nitroglycerin in the treatment of acute angina pectoris, characterized in that it comprises the steps of:

Detecting the ALDH2 gene, transcript and/or protein of the individual to be tested and comparing it to the normal ALDH2 gene, transcript and/or protein,

The difference indicates that nitroglycerin is less effective in treating acute angina pectoris than the general acute angina pectoris. '

9. The method of claim 8, wherein the gene or transcript of ALDH2 is detected and compared to a normal ALDH2 nucleotide sequence.

10. The method of claim 9, wherein the difference is a single nucleotide polymorphism:

1951 G→A;

Wherein, the nucleotide position number is based on SEQ ID NO: 1.