KR100504002B1 - Composition for diagnosis of polycystic ovary syndrome comprising primers detecting single nucleotide polymorphism at 677th nucleotide of 5,10-methylenetetrahydrofolate reductase gene - Google Patents

Abstract

The present invention relates to a composition for diagnosing polycystic follicle syndrome.

The composition of the present invention includes a primer set that can be used for PCR of a nucleotide sequence including 677 nucleotides of the MTHFR gene, and further comprises a sequence primer that can be used for pyro sequencing a PCR product obtained using the primer set. It may include.

The 677 C-> T SNP of MTHFR detectable by the composition of the present invention is a genetic indicator of the possibility of polycystic follicular syndrome.

Therefore, the composition of the present invention can be usefully used for diagnosis and treatment of polycystic follicle syndrome.

Description

Composition for diagnosis of polycystic ovary syndrome comprising primers detecting single nucleotide polymorphism at 677th nucleotide of 5 The primer for detecting a single base polymorphism at the nucleotide 677 of the methylenetetrahydrofolate reductase gene , 10-methylenetetrahydrofolate reductase gene}

The present invention relates to a composition for diagnosing polycystic follicle syndrome.

Polycystic follicle syndrome is a clinical syndrome showing polycystic follicle production, dysmenorrhea due to ovulation disorders, and masculinity.

Women with polycystic follicle syndrome often lead to infertility due to chronic ovulation, and are characterized by hyperandrogenism, obesity, hirsutism, hyperinsulinemia and insulin resistance, and diabetes, hypertension, arteriosclerosis, The risk of developing coronary artery disease, endometriosis, and lipid metabolism is 1-40% higher than normal.

Much of the cause of polycystic follicle syndrome is due to genetic abnormalities. Studies to identify genes related to polycystic follicle syndrome have been carried out to study gene expression related genes that regulate gonadotropin action, genes related to steroid hormone production regulation, genes related to insulin action and obesity and family history. It has been divided into the back. Once these genes are identified, accurate diagnosis and treatment of polycystic follicle syndrome will be possible.

However, the correlation between specific genes and polycystic follicle syndrome is not well known, and thus, proper treatment considering accurate diagnosis and genetic abnormalities cannot be achieved.

Accordingly, the present invention invented a composition that enables the accurate diagnosis and specific treatment of specific infertility symptoms by detecting SNPs of MTHFR (5,10-methylenetetrahydrofolate reductase) genes involved in homocysteine metabolism.

In the present invention, by identifying the SNP of 677 nucleotides of the MTHFR gene, to provide a composition that enables the accurate diagnosis and treatment direction setting of polycystic follicular syndrome.

The present invention provides a composition for diagnosing polycystic follicle syndrome comprising a primer capable of detecting a single nucleotide polymorphism (SNP) at the nucleotide 677 position of the MTHFR (5,10-methylenetetrahydrofolate reductase) gene. to provide.

The composition of the present invention includes a primer set that can be used for PCR (polymerase chain reaction) of a nucleotide sequence including 677 nucleotides of the MTHFR gene.

The primer set included in the composition of the present invention is used to amplify a portion of the MTHFR gene to be used for pyrosequencing or restriction fragment length polymorphism (RFLP) analysis.

The forward primer of the primer set used for pyro sequencing specifically has the nucleotide sequence of SEQ ID NO: 1, the reverse primer specifically has the nucleotide sequence of SEQ ID NO: 2.

In the primer set used for the RFLP, the forward primer specifically has a nucleotide sequence of SEQ ID NO: 3, and the reverse primer specifically has a nucleotide sequence of SEQ ID NO: 4.

The composition of the present invention may further comprise a sequence primer that can be used for pyro sequencing the PCR product obtained using the primer set.

The sequence primer included in the composition of the present invention specifically has a nucleotide sequence of SEQ ID NO.

Primers in the composition of the present invention may be provided in a powder state or dissolved in distilled water or a suitable buffer to maintain stability. In addition, in order to increase the efficiency and convenience of the purification process for the subsequent experimental step, biotin (biotin) can be attached.

In addition to the primers, the composition of the present invention may be composed of components required for PCR reaction, pyro sequencing, and RFLP analysis, as necessary.

Specifically, the components required for the PCR reaction may be metal ion salts such as deoxynucleotide triphosphate (dNTP), heat resistant polymerase, magnesium chloride, and the like, and the components required for sequencing include dNTP and sequinase. The components required for RFLP may be DNA restriction enzymes such as Hinf1, buffers for enzyme reactions, and the like.

In addition, the composition of the present invention may be added to the tube to be used for mixing each component, instructions for describing how to use, if necessary.

In the present invention, the nucleotide 677 of the MTHFR gene of a woman suspected of polycystic follicle syndrome exhibits any SNP of cytosine / cytosine (C / C), cytosine / thymine (C / T), or thymine / thymine (T / T). It provides a way to check.

When identifying the SNP at position 677 of the MTHFR by the method of the present invention, one or more of pyro sequencing or RFLP method may be specifically used.

When using the pyro sequencing method, a base containing nucleotide 677 of MTHFR is prepared by using a DNA set isolated from a woman suspected of polycystic follicle syndrome and performing a PCR reaction using a primer set included in the composition of the present invention. Amplify a portion of the sequence.

After annealing the PCR product obtained above and the sequence primer included in the composition of the present invention, pyro sequencing was performed to determine whether SNP of 677 nucleotides occurred.

In the present invention, the primer set used in the PCR reaction is prepared to amplify about 50-100 nucleotides on the left and right on the basis of 677 nucleotides in order to increase the efficiency of PCR. Specifically, the forward primer has a nucleotide sequence of SEQ ID NO: 1, the reverse primer preferably has a nucleotide sequence of SEQ ID NO: 2.

In the present invention, the sequence primer used for pyro sequencing is spaced about 10 to 30 nucleotides in the 5 'direction of 677 nucleotides in the nucleotide sequence of the PCR product obtained using the primer set to increase the accuracy of sequencing. Select the sequence to be placed. Specifically, the sequence primer preferably has a nucleotide sequence of SEQ ID NO: 3.

When using the RFLP method, a DNA sequence isolated from a sample is used as a template, and a PCR reaction is performed using a primer set included in the composition of the present invention, thereby amplifying a portion of the nucleotide sequence including 677 nucleotides of MTHFR.

After the reaction by adding the appropriate DNA restriction enzyme (restriction enzyme) to the PCR product obtained above, and check the DNA cleavage.

Primer set used in the PCR reaction in the present invention should be able to confirm whether the DNA cleavage while increasing the efficiency of the PCR, using the one prepared to amplify about 200 nucleotides, including 677 nucleotides. Specifically, the forward primer has a nucleotide sequence of SEQ ID NO: 4, the reverse primer preferably has a nucleotide sequence of SEQ ID NO: 5.

In the present invention, the restriction enzyme used for DNA cleavage is preferably Hinf1. Fragments obtained from the normal genotype MTHFR do not have a region that Hinf1 can recognize, but fragments obtained from the MTHFR that had a 667 C-> T mutation generate a cleavage site that Hinf1 can recognize, and are decomposed into 175bp and 25bp. The occurrence of single nucleotide SNP can be confirmed.

In the present invention, in addition to the above method, conventional techniques used for SNP detection such as direct sequencing can be used.

Hereinafter, the correlation between the MTHFR C677T gene SNP and polycystic follicular syndrome will be described with reference to the MTHFR-related metabolic process shown in FIG. 1.

As shown in Figure 1, MTHFR converts 5,10-methylenetetrahydrofolate (5,10-methylenetetrahydrofolate: 5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF) Is an enzyme.

Since 5-methylTHF provides the methyl group necessary when homocysteine remethylates with methionine, 5-methylTHF is an important factor in maintaining homocysteine concentration in blood.

However, when the 677th nucleotide of MTHFR is converted from cytosine to thymine (hereinafter referred to as 'MTHFR C677T'), the corresponding amino acid is substituted from alanine to valine. The entire nucleotide sequence of MTHFR normal gene and of MTHFR C677T are shown in SEQ ID NO: 6, SEQ ID NO: 7, respectively.

The MTHFR C677T gene mutation results in a severe decrease in the function of the MTHFR enzyme, so that the person with the heterozygous mutation 677CT genotype reduces the function of the MRHFR enzyme by 32.4% compared to the person with the normal 677CC genotype, and is homozygous. In humans with the 677TT genotype of mutation, it is reduced by 64.4% (Chango A et al., The effect of 677C → T and 1298A → C mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects.Br J Nutr 2000; 83 (6): 593-596).

As such, when the function of the MTHFR enzyme is degraded by mutation, 5-methyleneTHF is accumulated, which affects the conversion of dUMP to dTMP during DNA synthesis. As a result, as the production of dTMP becomes active, dTMP stress is reduced, and in some diseases, the risk of development seems to be lowered.

In fact, SNPs at the MTHFR 1298 site in patients with recurrent spontaneous miscarriage showed that habitual miscarriage increased as the frequency of genetic mutations decreased (Nam Kim, Do Yeon, Kim Sun Hee 5,10-Methylenetetrahydrofolate Reductase (MTHFR C677T and A1298C)). , A study on the relevance of repeated natural heritage of genetic mutation, Korean Journal of Infertility 2002; 29: 215-22).

In the case of colorectal cancer and acute leukemia, the incidence of C677T and A1298C mutations was rather low (Skibola CF et al., Polymorphism in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults.Proc Natl Acad Sci USA 96 : 12810-5, 1999; Chen J. et al., A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer.Cancer Res 56: 4862-4, 1996; Wiemels JL et al., MF.Methylenetetrahydrofolate reductase (MTHFR) polymorphism and risk of molecularly defined subtypes of childhood acute leukemia.Proc Nalt Acad Sci 98 (7): 4004-9, 2001).

Similarly, if the incidence of C677T SNP in the MTHFR gene is low compared to normal, the likelihood of polycystic follicular syndrome is high. In particular, the lower the frequency of homozygous mutations (T / T) and the T allele at position 677 of the MTHFR gene, the more likely the occurrence of polycystic follicular syndrome.

Thus, the compositions of the present invention allow for the prediction and diagnosis of polycystic follicular syndrome occurrence through SNP analysis of the 677 position of MTHFR.

In addition, as a result of using the composition of the present invention, a woman expected to exhibit polycystic follicle syndrome can be prevented or treated by administering a functional supplement of MTHFR mutant enzyme, so that the composition of the present invention is suitable for the treatment of polycystic follicular syndrome. This can be useful for establishing methods.

For example, using the composition of the present invention by reacting a test substance with a cell or animal identified to express the MTHFR 677 gene variant and confirming that the test substance lowers the homocysteine concentration, Functional supplements can be screened.

The search for a functional supplement of the MTHFR mutant may further lead to the development of a therapeutic agent for polycystic follicle syndrome.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

EXAMPLES Detection of MTHFR C677T Gene Variants and Diagnosis of Polycystic Follicle Syndrome by Compositions of the Invention

In order to detect MTHFR C677T gene variant by the composition of the present invention and to diagnose polycystic follicular syndrome, experiments were performed as follows.

1) Preparation of the composition of the present invention

The composition of the present invention was prepared to include two primer sets and sequence primers for pyrosequencing.

One set of primers is used for PCR for pyro sequencing, the base sequence of the forward primer is SEQ ID NO: 1, the base sequence of the reverse primer is SEQ ID NO: 2.

The other set of primers is used for PCR for RFLP analysis, the base sequence of the forward primer is SEQ ID NO: 3, the reverse sequence of the reverse primer is SEQ ID NO: 4.

The base sequence of the sequence primer is the same as SEQ ID NO: 5.

2) Test subject

Eighty-six patients diagnosed with polycystic follicular syndrome who visited Kangbuk Samsung Hospital and Pocheon Jung-Mun Medical School, and 100 control women who had children with normal natural pregnancy were included.

3) Pyro Sequencing Analysis

DNA for pyrosequencing analysis was prepared as follows.

DNA isolated from leukocytes obtained from each individual was amplified in a PCR machine (MJ research thermal cycler, Waltham, USA) using a PCR primer set (SEQ ID NO: 1, SEQ ID NO: 2) for pyro sequencing.

The procedure was repeated for 45 cycles with annealing at 55 ° C. to amplify the product at position 677 to 113 bp. The products were electrophoresed on a 2% agarose gel, followed by EtBr (ethidium). staining with bromide) to observe the amplification status.

PCR products with biotin attached were immobilized to superparamagnetic beads (streptavidin) with superparamagnetic beads (Dynabeads M280; Dynal, Oslo, Norway). PCR products immobilized in sodium hydroxide solution were incubated to obtain single-stranded DNA, followed by neutralization by addition of 0.2 M hydrochloric acid.

In order to perform SNP analysis on the single-stranded DNA thus extracted, pyro sequencing was performed using the composition of the present invention as follows.

10 pmol of the sequence primer (SEQ ID NO: 5) of the composition of the present invention was added to the single-stranded DNA to react at room temperature for 1 minute and at 80 ° C. for 2 minutes, and then slowly cooled to room temperature to anneal.

To the sequence primers anneal DNA, exonuclease I devoid of Klenow DNA polymerase ^{(exonuclease deficient (exo -) Klenow} DNA polymerase), Bahia cyclase (apyrase), purified luciferase (purified luciferase), recombinant ATP Sulfur Relabin (recombinant ATP sulfurylase), substrate and dNTP were added and pyro sequencing was performed at room temperature using an automated pyro sequencing device (kindly supplied by Pyrosequencing AB, Uppsala, Sweden).

At this time, the dNTP was added sequentially to extend the sequence primer, and the remaining nucleotides were removed by the apiase. Fluorescence signals emitted from the bound nucleotides were detected by a CCD camera mounted on a sequencing device.

4) RFLP Analysis

DNA to be used for RFLP analysis was prepared as follows.

PCR primer set for RFLP (SEQ ID NO: 3, SEQ ID NO: 4) was added to each group of DNA, denatured at 95 ° C., annealed the primer at 64 ° C. for 1 minute, and primer extension at 72 ° C. for 2 minutes. The procedure was repeated 30 times to obtain a PCR product of 198bp size.

For the PCR products, the restriction enzyme Hinf1 (New England Biolabs, Beverly, MA, USA) was added to perform degradation reaction at 37 ° C. for 4 hours. The fragments treated with restriction enzymes were electrophoresed on 3% agarose gel and stained with EtBr (ethidium bromide) to observe the mutated state.

The pyro sequencing results of each variation obtained by the above experiments and the corresponding RFLP results are shown in FIG. 2.

FIG. 2A shows the results of pyro sequencing of the normal genotype (C / C) and the mutations (C / T, T / T) at position 677 of MTHFR, and FIG. / C) and the variation (C / T, T / T) RFLP check results.

5) Statistical analysis of the results

For statistical analysis according to the classification method of each group, chi-square test and Fisher's exact test were performed using SAS statistical software. In addition, the odds ratio (OR) and 95% confidence interval (95% CI) were used to measure the comparative risk in polycystic follicular syndrome.

The results obtained by statistical analysis are as follows.

5-1) Overall MTHFR Genotype Distribution in Control and Patient Groups

Overall genotype distribution patterns of the control and patient groups are shown in Table 1 below.

Genotype and Classification Normal control group (%) PCO patient population (%) OR (95% CI) P 677CC 27.00 (n = 27) 38.37 (n = 33) - - 677CT 52.00 (n = 52) 53.49 (n = 46) - - 677TT 21.00 (n = 21) 8.14 (n = 7) 0.2727 (1.10-0.74) 0.0085 CT + TT 73.00 (n = 73) 61.63 (n = 53) - - T allele 47.00 (n = 47) 34.88 (n = 60) 0.6041 (0.40-0.91) 0.0180 1298AA 67.00 (n = 67) 61.63 (n = 53) - - 1298AC 28.00 (n = 28) 30.23 (n = 26) - - 1298CC 5.00 (n = 5) 8.14 (n = 7) - - AC + CC 33.00 (n = 33) 38.37 (n = 33) - - C allele 19.00 (n = 19) 23.26 (n = 40) - -

As shown in Table 1, the MTHFR genotype in the control group was 27.0% normal (C / C), 52.0% heterozygous (C / T), 21.0% homozygous (T / T), heterozygous + homozygous The frequency of (CT + TT) 73.0% and T allele 47.0%.

On the other hand, patients with polycystic follicular syndrome were 38.4.0% normal (C / C), 53.5% heterozygous (C / T), 8.14% homozygous (T / T), heterozygous + homozygous (CT + TT). ) 61.6%, T allele (34.9%).

In other words, in patients with polycystic follicular syndrome, the frequency of homozygous mutations and T alleles at position 677 was significantly reduced compared to the normal control group (homozygous mutations: P = 0.0085, T allele: P = 0.0180). It was also reduced in both cases.

On the other hand, the mutation of position 1298, another common genetic variation of MTHFR, showed no statistical difference between the control group and the patient group, unlike the mutation of position 677 (P> 0.05).

In the above, it can be seen that the occurrence of polycystic follicle syndrome is closely related to the frequency of SNP occurrence at the MTHFR 677 position.

Thus, the compositions of the present invention which detect SNPs at the MTHFR 677 position can be used for the diagnosis of polycystic follicle syndrome, as well as for the prevention and early treatment.

Of the SNPs of MTHFR detectable by the compositions of the present invention, the frequency of 677 C-> T mutations is a genetic indicator of the likelihood of developing polycystic follicular syndrome.

Therefore, the composition of the present invention can be effectively used for the diagnosis of polycystic follicular syndrome.

In addition, when the composition of the present invention is found to be polycystic follicle syndrome, the above-described symptoms can be prevented or treated by administering a functional supplement of MTHFR mutant to the patient, the composition of the present invention is suitable for the treatment direction of polycystic follicle syndrome This can be useful for determining

1 is a diagram showing metabolic pathways involved in MTHFR.

Figure 2 shows the results of confirming the genotype of the normal group and polycystic follicular syndrome patient group through pyro sequencing and RFLP analysis.

<110> LEE, Su Man <120> Composition for diagnosis of polycystic ovary syndrome comprising primers detecting single nucleotide polymorphism at 677th nucleotide of 5,10-methylenetetrahydrofolate reductase gene <130> 03P-23 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward PCR primer amplifying the part of methylenetetrahydrofolate reductase for pyrosequencing <400> 1 tattggcagg ttaccccaaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse PCR primer amplifying the part of methylenetetrahydrofolate reductase for pyrosequencing <400> 2 gaaaagctgc gtgatgatga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward PCR primer amplifying the part of methylenetetrahydrofolate reductase for RFLP analysis <400> 3 tgaaggagaa ggtgtctgcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse PCR primer amplifying the part of methylenetetrahydrofolate reductase for RFLP analysis <400> 4 aggacggtgc ggtgagagtg 20 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> primer for pyrosequencing <400> 5 ggtgtctgcg gga 13 <210> 6 <211> 302 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (302) <223> human methylenetetrahydrofolate reductase wild type gene <400> 6 ccttgaacag gtggaggcca gcctctcctg actgtcatcc ctattggcag gttaccccaa 60 aggccacccc gaagcaggga gctttgaggc tgacctgaag cacttgaagg agaaggtgtc 120 tgcgggagcc gatttcatca tcacgcagct tttctttgag gctgacacat tcttccgctt 180 tgtgaaggca tgcaccgaca tgggcatcac ttgccccatc gtccccggga tctttcccat 240 ccaggtgagg ggcccaggag agcccataag ctccctccac cccactctca ccgcaccgtc 300 ct 302 <210> 7 <211> 302 <212> DNA <213> Homo sapiens <220> <221> mutation (222) (1) .. (302) <223> human methylenetetrahydrofolate reductase C667T mutant gene <400> 7 ccttgaacag gtggaggcca gcctctcctg actgtcatcc ctattggcag gttaccccaa 60 aggccacccc gaagcaggga gctttgaggc tgacctgaag cacttgaagg agaaggtgtc 120 tgcgggagtc gatttcatca tcacgcagct tttctttgag gctgacacat tcttccgctt 180 tgtgaaggca tgcaccgaca tgggcatcac ttgccccatc gtccccggga tctttcccat 240 ccaggtgagg ggcccaggag agcccataag ctccctccac cccactctca ccgcaccgtc 300 ct 302

Claims (14)

A primer set capable of detecting SNP at 677 nucleotide position of MTHFR (5,10-methylenetetrahydrofola1te reductase) gene. A forward primer has a nucleotide sequence of SEQ ID NO. 1, and a reverse primer has a nucleotide sequence of SEQ ID NO. Polycystic follicle syndrome diagnostic composition comprising a set delete delete delete The composition for diagnosing polycystic follicle syndrome according to claim 1, further comprising a sequence primer that can be used for pyrosequencing of a PCR product obtained using the primer set. 6. The composition for diagnosing polycystic follicle syndrome according to claim 5, wherein the sequence primer has a nucleotide sequence of SEQ ID NO: 5. delete delete Identifying a SNP of MTHFR 677 nucleotide consisting of the following steps; (1) first step of isolating MTHFR DNA from a woman suspected of polycystic follicle syndrome; (2) a second method of performing a PCR reaction using the isolated MTHFR DNA as a template, the forward primer having the nucleotide sequence of SEQ ID NO: 1, and the reverse primer using the PCR primer set having the nucleotide sequence of SEQ ID NO: 2; step; (3) a third step of annealing the PCR product and the sequence primer and performing pyro sequencing; And (4) nucleotide 677 of the MTHFR gene of a woman suspected of having polycystic follicle syndrome has any SNP of cytosine / cytosine (C / C), cytosine / thymine (C / T), or thymine / thymine (T / T). 4th step to check if 10. The method of claim 9, wherein the sequence primer has a nucleotide sequence of SEQ ID NO: 5 method for identifying the SNP of nucleotide MTHFR 677 delete delete delete delete