KR100504004B1 - Composition for diagnosis of male infertility 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 male infertility.

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 SNPs of MTHFR detectable by the compositions of the present invention are genetic indicators indicating the likelihood of unexplained atherosclerosis and severe rare atresia.

Therefore, the composition of the present invention can be usefully used for setting the direction of diagnosis and treatment of infertility diseases.

Description

Composition for diagnosis of male infertility comprising primers detecting single nucleotide polymorphism at 677th nucleotide of 5,10 -methylenetetrahydrofolate reductase gene}

The present invention relates to a composition for diagnosing male infertility.

Male infertility has a wide variety of symptoms, causes, and prognosis, making it difficult to accurately diagnose and treat.

According to the World Health Organization (WHO), male infertility is characterized by azoospermia, oligospermia, astheno spermia, tetrazoospermia, and a combination of symptoms. According to the incidence and severity, various clinical symptoms such as oligoasthenotetrazoospermia (OAT) and severe Severe OAT are shown.Indeed, the most frequent symptoms are spermatosis, rarely deformed spermosis, and severe It is summarized as rare armed anomaly.

Aspermatosis refers to a condition in which no sperm is present in the ejaculate.

Rare anomaly spermosis occurs when the amount of sperm is less than 20 * 10 ⁶ , the motility is less than 50% than normal, and the sperm malformation is 4% or more. .

Severe rare dysmorphic spermosis is diagnosed when the sperm has less than 20 * 10 ⁶ sperm and the motility and morphology are lower than that of the common rare disorder.

Male infertility, which is caused by a variety of symptoms, has various causes, and infertility due to genetic aberration is known to account for more than 30% of the total.

The human genome contains hundreds of thousands of genes, of which about 1,000 genes are known to regulate human reproduction. Monogene mutations can cause reproductive problems, while germline development, gonad development and physical development in men are regulated by complex genetic networks.

Genetic abnormalities occur during spermatogenesis in humans and, to date, are known as numerical or structural abnormalities of chromosomes.

The most common numerical abnormality of chromosomes in infertile men is Klinefelter syndrome, which is characterized by 47, XXY, or 46, XY / 47XXY in mosaic form, with non-chromosome segregation during mitosis of the X chromosome. caused by disjunction), characterized by small testicle size and spermatosis. On the other hand, structural abnormalities of chromosomes include damage to chromosomes such as deletion, duplication and inversion.

Partial deletion of the Y chromosome (Y-chromosome microdeletion) also accounts for many causes of male infertility.

The Y chromosome consists of three parts, the chromosomal portion of the forearm (Yp11), the chromosomal portion of the forearm (Yq11), and the heterochromatic portion of the forearm (Yq12), which contain genes that perform different functions in male reproduction. Doing. Infertility also depends on which part of the Y chromosome has been deleted.

For example, microdeletion of the DAZ (Deleted in Azoospermia) gene occurs at the AZF (azoospermic factor) position of the Y chromosome of the Y chromosome. In addition, AZFa-deleted patients do not have germ cells, and AZFb-deleted patients show mutational suppression or meiosis inhibition before spermatogenesis, and deletions in AZFc are associated with atherosclerosis or severe rare spermosis. .

As such, male infertility is caused by a variety of genetic causes, the genetic mechanism that causes a particular infertility symptoms must be revealed to accurately diagnose and treat the infertility symptoms.

However, apart from chromosomal aberrations or deletions, the correlation between specific genes and specific infertility symptoms is not well known. Therefore, at present, diagnosis and treatment of infertility can only be performed by general criteria without considering the genetic characteristics of specific infertility symptoms. Indeed, a significant proportion of male infertility, rare armed deformity spermosis, severe rare deformity spermosis is often diagnosed as unknown cause, the situation is not adequate treatment.

Therefore, in the present invention, MTHFR 677 mutation genotype involved in homocysteine metabolism is associated with general male infertility (Bezord et al., Homozygous Methylenetetrahydrofolate Reductase C677T Mutation and Male Infertility.N Engl J Med 15: 1172; 2001) By inventing the SNPs of the genes, a composition has been invented that enables accurate diagnosis and setting of treatment directions for specific infertility symptoms.

In the present invention, by identifying the SNP of 677 nucleotides of the MTHFR gene, it is intended to provide a composition that enables the accurate diagnosis and treatment direction of cause unknown spermatosis and severe rare atypical spermatosis.

The present invention provides a composition for diagnosing male infertility comprising a primer capable of detecting a single nucleotide polymorphism (SNP) at a nucleotide 677 position of a MTHFR (5,10-methylenetetrahydrofolate reductase) gene. do.

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.

Male infertility that the compositions of the present invention can be diagnosed is either unexplained atherosclerosis or severe rare atresia of etiology.

The present invention is to determine whether the 677 nucleotides of the MTHFR gene of male suspected of infertility, which SNP of cytosine / cytosine (C / C), cytosine / thymine (C / T), or thymine / thymine (T / T) Provide a way to.

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 portion of the nucleotide sequence containing 677 nucleotides of MTHFR is amplified by PCR using a primer set included in the composition of the present invention as a template using DNA isolated from infertility males. .

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 unknown causative atherosclerosis and severe rare dysmorphic spermosis 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, the degradation of MTHFR enzyme causes 5-methyleneTHF to accumulate more than necessary, affecting the conversion of dUMP to dTMP during DNA synthesis. As a result, overproduction of dTMP leads to aberration of DNA synthesis, which may be related to male infertility.

In addition, due to the mutation of the MTHFR 677 gene, a disorder of remethylation occurs during the conversion of homocysteine to methionine in the metabolic process shown in FIG. 1, thereby inhibiting the process, thereby resulting in high homocysteineemia (hyperhomocysteinemia). Will cause.

In particular, the homozygous mutant genotype (MTHFR 677TT) has a stronger effect on the accumulation of homocysteine, and excessive increase in the amount of homocysteine is likely to cause intestinal vascular wall damage and infertility.

As confirmed in the Examples of the present invention, C677T SNP of MTHFR has no correlation with male infertility due to chromosomal numerical, structural abnormality and Y chromosome partial deletion, whereas acute or severe among subtypes of unknown male infertility It is most commonly seen in male patients with rare disabilities.

Occurrence of unexplained atherosclerosis includes heterozygous mutations (C / T) and homozygous mutations (T / T) at the MTHFR 677 position, and the occurrence of severe rare dysmorphic spermatosis of unknown cause (I / O). / T) is closely related.

Thus, the compositions of the present invention allow for the prediction and diagnosis of unknown causative atherosclerosis and severe rare atypical spermosis through SNP analysis at position 677 of MTHFR.

That is, in the existing infertility diagnosis test, because it was difficult to diagnose infertile male infertility, the scope of diagnosis was limited to male infertility due to chromosomal abnormalities and partial deletion of Y chromosome, Finding out the causative factors of severe rare armed spermosis enables accurate genetic diagnosis.

In addition, as a result of using the composition of the present invention, it is possible to prevent or treat the above symptoms by administering a functional supplement of MTHFR mutase to a man expected to exhibit the infertility symptoms, the composition of the present invention according to various subtypes of male infertility It can be useful to establish proper treatment.

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 supplements of MTHFR mutant enzymes can further lead to the development of therapeutic agents for unknown spermatosis or severe rare atresia.

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 Composition of Unexplained Amorphism and Serious Rare Atypical Spermosis by Compositions of the Present Invention

In order to detect the MTHFR C677T gene variant by the composition of the present invention, and to diagnose unidentified atherosclerosis and severe rare asthma malformation, 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

The subjects were 377 male infertility patients who agreed to the study among men diagnosed with infertility at Gangnam CHA Hospital, Pocheon College of Medicine, and 396 control groups who had more than one child among normal people.

For infertile patients, cytogenetic analysis and Y-chromosome deletion analysis were performed as follows to diagnose the cause of infertility.

Cytogenetic analysis was performed when lymphocytes were cultured and spread into metaphase. DNA was isolated from leukocytes extracted from peripheral blood of patients and controls, and also one SRY and 12 Sequence Tagged Sites (STSs) (AZF-a region: Sy 84, sY86, AZF-b region: sY 134, sY 138, MK5, AZF-c region: 13 positions containing sY 152, sY 147, sY 254, sY 255, sPGY1, sY 269, sY 158) were subjected to multiple PCR (multi-PCR) using primers of the Y chromosome By amplification.

As a result, the infertility group was divided into two groups (115 people) and unknown groups (262).

The infertility group of unknown cause was classified into four subtypes according to the diagnosis name: 281 adolescents, 59 severe schizophrenia, 26 rare spermatozoa, 26 spontaneous abortion, spontaneous abortion, and retractile testis. testis, severe severe oligo sperm, and difficulty in ejaculation.

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 normal genotype (C / C) and mutations (C / T, T / T) at position 677 of MTHFR, and FIG. 2B shows normal genotype (C / C) in the normal control and infertile patients, respectively. ) And the result of RFLP check for the variation (C / T, T / T).

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 percent confidence intervals (95% CI) were used to measure the comparative risk in male infertility.

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 (%) Infertility group (%) OR (95% CI) P 677CC 36.62 28.12 - - 677CT 50.50 55.17 1.42 (1.03-1.95) 0.0290 677TT 12.88 16.71 1.69 (1.08-2.64) 0.0207 CT + TT 63.38 71.88 1.48 (1.09-2.00) 0.0117 T allele 38.13 44.30 1.29 (1.05-1.58) 0.0138 1298AA 67.93 63.40 - - 1298AC 28.03 31.83 - - 1298CC 4.04 4.77 - - AC + CC 32.07 36.60 - - C allele 18.06 20.69 - -

As shown in Table 1, the MTHFR genotype in the control group showed a frequency of 36.6% normal (C / C), 50.5% heterozygous (C / T), and 12.9% homozygous (T / T).

In the patient group, the normal (C / C) genotype was only 28.1%, and the variant genotype accounted for 55.2% of heterozygous mutations (C / T) and 16.71% of homozygous mutations (T / T). (P <0.05).

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).

Therefore, it can be seen that among the SNPs of MTHFR, the C677T mutation is closely related to male infertility.

5-2) Distribution of MTHFR Genotypes according to Symptom Subtypes in Infertile Patients

When the infertile patients were classified into three subtypes of symptom-type subtypes, namely, spermatosis, rarely deformed spermosis, and severe rare deformity spermosis, the MTHFR genotype frequency of each subtype is shown in Table 2 below.

Classification 전자 Genotype 677CC 677CT 677TT CT + TT T allele Normal control 36.62% (n = 145) 50.50% (n = 200) 12.88% (n = 51) 63.38% (n = 251) 38.13% (n = 302) Autism (n = 281) OR (95% CI) P 26.33% (n = 74)- 56.94% (n = 160) 1.57 (1.10-2.22) 0.0113 16.73% (n = 47) 1.81 (1.11-2.93) 0.0164 73.76% (n = 207) 1.62 (1.16-2.26) 0.0048 45.20% (n = 254) 1.34 (1.07-1.67) 0.0092 severe OAT (n = 59) OR (95% CI) P 25.42% (n = 15)- 50.85% (n = 30)- 23.73% (n = 14) 2.65 (1.20-5.88) 0.0136 74.58% (n = 44)- 49.15% (n = 58) 1.57 (1.06-2.31) 0.0224 OAT (n = 26) OR (95% CI) P 50.00% (n = 13)- 38.46% (n = 10)- 11.54% (n = 3)- 50.00% (n = 13)- 30.77% (n = 16)- Remaining patient group (n = 11) OR (95% CI) P 36.36% (n = 4)- 63.64% (n = 7)- - - 63.64% (n = 7)- 31.82% (n = 7)-

As shown in Table 2, there was a statistically significant genetic variation in the MTHFR 667 position in the sperm patients group and in the patients with severe rare atherosclerosis.

In the group of patients with atypical disorder, 56.9% of heterozygous mutations (C / T), 16.7% of homozygous mutations (T / T), 73.7% of combined heterozygosity and homozygous (CT + TT), and 45.2% of T allele (allele) Frequency was shown and statistically significant (P <0.05).

In the patients with severe rare atresia, the frequency of homozygous mutation (T / T) was 16.73% and the T allele was 49.2% with statistical significance (P <0.05). In particular, the comparative risks of patients with severe rare malformations were significantly higher (OR: 2.65, 95% CI: 1.20-5.88).

5-3) Distribution of MTHFR Genotypes According to Symptom Subtypes in Unexplained Infertile Patients

MTHFR genotype distribution was analyzed by classifying infertile patients into known and unknown causes of chromosomal abnormalities.

In the case of infertility group due to chromosomal abnormality, no significant result was observed for the mutation, and therefore, only the statistical analysis results for the unknown infertility group are shown in Table 3 below.

Classification 전자 Genotype 677CC 677CT 677TT CT + TT T allele Normal control 36.62% (n = 145) 50.50% (n = 200) 12.88% (n = 51) 63.38% (n = 251) 38.13% (n = 302) Autism (n = 281) OR (95% CI) P 25.66% (n = 49)- 56.54% (n = 108) 1.60 (1.07-2.38) 0.0210 17.80% (n = 34) 1.97 (1.15-3.39) 0.0132 74.35% (n = 142) 1.67 (1.14-2.46) 0.00482 46.07% (n = 176) 1.39 (1.08-1.77) 0.0095 severe OAT (n = 59) OR (95% CI) P 21.95% (n = 9)- 53.66% (n = 22)- 24.39% (n = 10) 3.16 (1.22-8.21) 0.0140 78.05% (n = 32) 2.05 (0.95-4.42) 0.0613 51.22% (n = 42) 1.70 (1.08-2.69) 0.0209 OAT (n = 26) OR (95% CI) P 54.17% (n = 13)- 33.33% (n = 8)- 12.50% (n = 3)- 45.83% (n = 13)- 29.17% (n = 14)- Remaining patient group (n = 11) OR (95% CI) P 50.00% (n = 3)- 50.00% (n = 3)- - - 50.00% (n = 3)- 25.00% (n = 3)-

The results of Table 3 showed a similar aspect to Table 2.

The group of patients with atherosclerosis had a heterozygous mutation (C / T) of 56.54%, a homozygous mutation (T / T) of 17.80%, a heterozygous and homozygous combination of 74.35%, and a T allele of 46.07%. Frequency was shown and statistically significant (P <0.05).

Patients with severe rare atypical sperm disease had a frequency of 53.66% homozygous mutation (T / T) and 51.22% T allele (OR: 3.16, 95%). CI: 1.22-8.21) and high significance (P = 0.0140).

In the above, it can be seen that the incidence of unknown etiology is closely related to heterozygous mutation (C / T) (P = 0.02) and homozygous mutation (T / T) (P = 0.01) at the MTHFR 677 position.

In addition, in cases of severe, rare, atypical spermatozoa, the homozygous mutation (T / T) at the MTHFR 677 position may be an important genetic feature.

Accordingly, the compositions of the present invention which detect SNPs at the MTHFR 677 position can be used for the diagnosis of unexplained atherosclerosis or severe rare atresia, and for the prevention and early treatment.

Among the SNPs of MTHFR that can be detected by the compositions of the present invention, the 677 C-> T mutation is a genetic indicator indicating the likelihood of unexplained atherosclerosis and severe rare atypical spermosis, which account for a large proportion of male infertility.

Therefore, the composition of the present invention can be effectively used for the diagnosis of male infertility.

In addition, the composition of the present invention is useful for determining the appropriate treatment direction of infertility when it is determined that the infertility by the composition of the present invention, by administering a functional supplement of MTHFR mutase to the subject patient. Can be used.

1 is a diagram showing metabolic pathways involved in MTHFR.

Figure 2 shows the results of confirming the genotype of the normal group and male infertility patients through pyro sequencing and RFLP analysis.

<110> LEE, Su Man <120> Composition for diagnosis of male infertility comprising primers detecting single nucleotide polymorphism at 677th nucleotide of 5,10-methylenetetrahydrofolate reductase gene <130> 03P-22 <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 (16)

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. Male infertility diagnostic composition comprising the set delete delete delete delete The composition for diagnosing male infertility according to claim 1, further comprising a sequence primer having a nucleotide sequence of SEQ ID NO: 5 that can be used for pyrosequencing of a PCR product obtained using the primer set. The composition for diagnosing male infertility according to claim 6, wherein the male infertility is azoospermia. 7. The composition for diagnosing male infertility according to claim 6, wherein the male infertility is severe oligoas the notetrazoospermia of unknown cause. delete Identifying a SNP of MTHFR 677 nucleotide consisting of the following steps;  (1) the first step of separating MTHFR DNA from infertile men; (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) a nucleotide number 677 of the MTHFR gene of infertility male to indicate which SNP of cytosine / cytosine (C / C), cytosine / thymine (C / T), or thymine / thymine (T / T) 4 steps delete The method of claim 10, wherein the sequence primer has a nucleotide sequence of SEQ ID NO: 5 method for identifying the SNP of the nucleotide MTHFR 677 delete delete delete The function of the MTHFR mutant enzyme, characterized by reacting the test substance with cells discharged in vitro from MTHFR-expressing cells having a genetic mutation at 677, and confirming that the test substance lowers the homocysteine concentration in blood. Complement Screening Method