KR100909372B1 - Methods for diagnosing premature ovarian failure using polynucleotides comprising single nucleotide polymorphism - Google Patents

Abstract

The present invention provides a polynucleotide or a complementary polynucleotide thereof including Single-Nucleotide Polymorphism (SNP) derived from BCKDHB (Branched Chain Keto acid DeHydrogenase E1, Beta polypeptide) gene. Complementary polynucleotides can be used as markers for clinical diagnosis of Premature Ovarian Failure (POF).

Early Menopause, Monobasic, BCKDHB

Description

Methods for diagnosing early menopause using polynucleotides containing single nucleotide polymorphisms {Methods for diagnosing premature ovarian failure using polynucleotides comprising single nucleotide polymorphism}

The present invention provides polynucleotides or their complementary nucleotides, including monobasic polymorphisms associated with premature menopause; An amplification primer or early menopause diagnostic probe for early menopause diagnosis comprising the polynucleotide or its complementary nucleotides; A microarray comprising the probe; And it relates to a method for early menopause diagnostic using the polynucleotide or its complementary nucleotides.

The human genome consists of 22 pairs of homologous chromosomes and two sex chromosomes. But every human being has a different appearance and character. This is because each person has the same number of chromosomes, but all of the phenotypes of the genes expressed from that chromosome are different. The diversity of these gene expressions in individuals is due to the genetic diversity of the genome carrying the gene. Much research has been done on this genetic diversity, and the structure of the genetic genome has recently been revealed.

Genetic diversity in the genome is known as transposable elements, short tandem repeats (STRs), variable number tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs). Dual SNPs are monobasic, appearing at a frequency of about one thousand nucleotides on the human genome and taking the form of single nucleotide variations between individuals of the same species. The genetic meaning of these monobasic polymorphisms makes a big difference in each individual depending on its location. For example, when a monobasic polymorph is present at a location that encodes a protein, it may affect the structure of the protein, altering protein function and associated with disease. In another example, if a monobasic polymorph is present on another gene that does not encode a protein, ie, on a promoter or intron, the expression level of the protein may differ for each, resulting in a decrease in the overall activity of the protein. In addition, a protein may be abnormally expressed through alterative splicing.

Conventional techniques for the discovery of such monobasic polymorphisms include Restriction Fragment Length Polymorphism (RFLP) using restriction enzymes, TaqMan ^™ probe method using Allele-specific hybridization, and melting temperature ( dynamic allele-specific hybridization (DASH) using melting temperature (Tm) and pyrosequencing using polymerization. Recently, microarray technology integrated probes using the principle of Allele-Specific Extension onto small substrates, planted them, hybridized with target DNAs, and extended them to find a single base polymorphism. have.

On the other hand, as the woman ages, the function of the ovary is stopped and estrogen production is reduced and menstruation is stopped, which is called menopause. Normal menopause occurs between about 42 and 56 years of age, but early menopause, or early ovarian failure, occurs in about 1% of women. Therefore, if a single nucleotide polymorphism is identified that may be specific to early menopause patients, early menopause may be diagnosed.

Currently, several monobasic polymorphic sites have been identified in Follicle Stimulating Hormone Receptor (FSHR) and Inhibin alpha subunit (INHα). However, most of these are used to confirm the genotype of patients already diagnosed with early menopause, and does not show a significant difference from the control group. Thus, there is a need in the art to find new monobasic polymorphic sites associated with premature menopause.

The present inventors conducted a study to find a single nucleotide polymorphism specific to early menopause in women, especially Korean women, and thus, the BCKDHB (Branched Chain Keto acid DeHydrogenase, E1-beta subunit) gene was specific to premenopausal patients. The present invention was completed by finding a single base polymorphic site.

Accordingly, it is an object of the present invention to provide a polynucleotide or its complementary polynucleotide comprising a monobasic polymorphism associated with premature menopause.

It is also an object of the present invention to provide an amplification primer or early menopause diagnostic probe for early menopause diagnosis consisting of the polynucleotide or its complementary nucleotides.

Another object of the present invention is to provide a microarray including the probe.

In addition, an object of the present invention is to provide an early menopause diagnostic method using the polynucleotide or its complementary nucleotides.

According to an aspect of the present invention, a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NOs: 1 to 10 using primers or probes whose template is a polynucleotide sequence selected from the group consisting of polynucleotides of SEQ ID NOs: 1 to 10 A method for analyzing a monobasic polymorphism, wherein the monobasic polymorphism comprises at least 10 contiguous DNA sequences (hereinafter referred to as "polynucleotide (a)") comprising the 201 base of the DNA sequence of SEQ ID NO: 1, SEQ ID NO: 10 or more contiguous DNA sequences comprising the 201 base of the DNA sequence of 2 (hereinafter referred to as "polynucleotide (b)"), 10 or more contiguous sequences comprising the 131 th base of the DNA sequence of SEQ ID NO: 3 10 or more contiguous DNA sequences comprising the DNA sequence (hereinafter referred to as "polynucleotide (c)"), 201 base of the DNA sequence of SEQ ID NO: 4 (hereinafter, "Polynucleotide (d)"), at least 10 contiguous DNA sequences comprising the 201 base of the DNA sequence of SEQ ID NO: 5 (hereinafter referred to as "polynucleotide (e)"), DNA of SEQ ID NO: 6 10 or more contiguous DNA sequences comprising the 805 th base of the sequence (hereinafter referred to as "polynucleotide (f)"), 10 or more contiguous DNA sequences comprising the 201 base of the DNA sequence of SEQ ID NO: Hereinafter referred to as "polynucleotide (g)", 10 or more consecutive DNA sequences comprising the 201 base of the DNA sequence of SEQ ID NO: 8 (hereinafter referred to as "polynucleotide (h)"), SEQ ID NO: 9 10 or more contiguous DNA sequences comprising the 201 base of the DNA sequence of the following (hereinafter referred to as "polynucleotide (i)") and 10 or more contiguous DNA comprising the 501st base of the DNA sequence of SEQ ID NO: 10 DNA sequence selected from the group consisting of sequences (hereinafter referred to as "polynucleotide (j)") That is composed of as claimed, a method for analyzing a single nucleotide polymorphism of a polynucleotide selected from the group consisting of a polynucleotide of SEQ ID NO 1 to 10 is provided.

In addition, according to another aspect of the present invention, there is provided a method for early menopause diagnosis using the polynucleotide.

One or more polynucleotides selected from the group consisting of polynucleotides (a) to (j) according to the present invention or complementary nucleotides thereof may be usefully used for the diagnosis of early menopause.

In addition, primers or probes consisting of the polynucleotide or its complementary nucleotides, and the polynucleotide or the complementary nucleotide kit thereof may be usefully used for the diagnosis of early menopause.

In addition, the diagnostic method of the present invention can effectively diagnose early menopause.

The present invention provides polynucleotides or complementary nucleotides thereof selected from the group consisting of polynucleotides (a) to (j) associated with premature menopause.

The DNA sequences of SEQ ID NOs: 1 to 10 are monobasic polymorphisms showing a statistically significant difference (significance level p value <0.05) in the premenopausal patient group and the normal group, and they are BCKDHB (Branched Chain Keto acid DeHydrogenase, E1-beta). subunit). BCKDHB is an enzyme that acts on the amino acid breakdown of proteins, and it is surprising that the monobasic polymorphism is associated with premature menopause.

The present invention includes a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) or a complementary nucleotide thereof, and the length of the polynucleotide or the complementary nucleotide thereof is 10 to 100. Nucleotides, preferably 20 to 60 nucleotides, more preferably 40 to 60 nucleotides.

At least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) according to the present invention is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a monobasic polymorphism in a nucleotide sequence. A polymorphic site refers to a site where a monobasic polymorphism occurs in the polymorphic sequence. The polynucleotide can be DNA or RNA.

In the present invention, at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) is involved in early menopause. This was confirmed by analyzing the genotypes of alleles present in each monobasic polymorphism in DNA obtained from blood samples from premenopausal patients and normal individuals. In the present invention, the normal group and the patient group participated in 16 persons each, which was used for data analysis together with epidemiological investigations such as age and body mass index (BMI). The size of the cluster used for genotyping is shown in Table 1 below.

group Normal people Patient group total Survey 16 16 32

Tables 2a, 2b, and 3 below show the association of the DNA sequences of SEQ ID NOs: 1-10 with premature menopause and the characteristics of the polymorphic sequences.

In Table 2a and 2b, what each column means is as follows.

SNP represents a base observed at the site of a single polymorphism, where A1 and A2 are the mass alleles A1 and mass in the course of genotyping by Infinium ^TM Assay from Illumina. This small allele is named A2 (minor allele) arbitrarily for convenience of experiments.

Sequence number is a sequence number which represents each monobasic polymorphism.

The analysis was performed using Illumina's Infinium ^TM Assay method, which was performed with the consent of 32 volunteers in 16 cases and 16 controls each.

In the allele frequency column, cas_A2, con_A2 and Delta are the frequency of the A2 allele in the case sample, the frequency of the A2 allele in the normal sample, and the cas_A2 and the con_A2, respectively. It represents the absolute value of the difference. Since human chromosomes are inherited one from the father and one from the mother, they form a pair, and each allele is present in two pairs. Therefore, since one person has two alleles, the frequency of alleles is ‘1 person × 2’. Where cas_A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype) / (number of samples of disease group x2) in disease group, and con_A2 is (frequency of A2A2 genotype x2 + frequency of A1A2 genotype) / (normal group of disease group) Number of samples x2).

Genotype frequency represents the frequency of each genotype, and cas_A1A1, cas_A1A2 and cas_A2A2 and con_A1A1, con_A1A2 and con_A2A2 represent the number of humans with A1A1, A1A2 and A2A2 genotypes in the disease and normal groups, respectively.

Chi-square was used to analyze whether the frequency of the two alleles in the patient group and the normal group had a statistically significant difference. This is called a case-control study of alleles associated with the disease. Here, the significance level (rejection zone) was set to p_value <0.05, and it was judged that the frequency of alleles of each monobasic polymorphism in the patient group and the normal group showed a significant difference as p_value <0.05 in the chi-square test. In particular, the single base polymorphism corresponding to SEQ ID NO: 1 to 8 is expressed as p_value <0.01, which means that the difference is very significant, and can serve as a strong reference point for dividing the patient group from the normal group.

Here, chi-exact-p_value (chi-exact-p_value) is a value obtained from a chi-square distribution table having a degree of freedom (df) of 2. In the chi-square test, if the expected frequency is less than 5, the general chi-square test result may be inaccurate. Therefore, this variable is used to test statistical significance more accurately through Fisher's exact test.

The risk allele refers to an allele that occurs significantly more frequently in the patient group than the normal group among the two alleles, where A2 was selected in all 10 monobasic polymorphisms.

Odds ratio represents the ratio of the probability of having a risk allele among the normal group to the probability of having a risk allele among the patient group. In the present invention, the Mantel-Haenszel odds ratio method was used. CI represents the 95% confidence interval of the odds ratio and represents (lower confidence interval, upper confidence interval). Confidence intervals with a value of 1 cannot be considered significant for association with disease.

The HWE state represents the state of Hardy-Weinberg Equilibrium, and con_HWE and cas_HWE represent the state of Hardy-Wineberg equilibrium in normal and diseased groups. Based on chi-value = 3.84 (p-value = 0.05, df = 1) in the chi-square (df = 1) test, it is judged by Hardy-Weinberg equilibrium HWE (Hardy-Weinberg Equilibrium) if it is greater than 3.84, If less than 3.84, it was judged as Hardy-Weinberg Disequilibrium.

The meaning of each column in Table 3 is as follows. Here, columns such as chromosome, band, gene, description, SNP role, amino acid change, etc. are the itemized description of each monobasic polymorphism.

The marker name is a number added for convenience to the single base polymorph found.

Rs stands for Reference Sequence (RS), which is the name given after the Human Genome Project to identify each single nucleotide polymorphism in the database of the National Institute of Biotechnology Information (NCBI).

In chromosome, the number of chromosomes in which the single nucleotide polymorphisms are located.

The position column indicates the number of bases in which the single nucleotide polymorphisms are located when the entire nucleotide base constituting the chromosome 6 is numbered.

ㆍ Band shows the unit divided according to the color when the chromosome is dyed by a specific technique. This is called G-band.

Gene refers to a gene in which the single nucleotide polymorphisms are located, which is located in a gene called BCKDHB.

ㆍ Description is a description of the biochemical and metabolic activity of the gene BCKDHB, showing the main function of this gene. Based on this, you can find out how it affects the disease.

The SNP role indicates where the single base polymorphisms are located on the gene (BCKDHB). Thus, it can be seen how each monobasic polymorphism works in gene expression and function.

A change in amino acid is a description of whether the single nucleotide polymorphisms change the amino acids constituting the protein when a gene is expressed and a protein is generated, and here, all of the single nucleotide polymorphisms encode a protein. ), It is not related to amino acid changes.

The meaning of each column in Table 4 is as follows.

• The SNP shows the unique number and alleles of each monobasic polymorph.

Allele; OR (95% CI) and Allele; p-value indicate how risk alleles give the disease an odds ratio (OR) through the ratio of the frequency at which alleles appear between patients and normal subjects. The 95% confidence interval (CI) for this result is shown. The 95% confidence interval does not include the number 1, indicating that the results are significant, and the significance level in the population (p-value = 0.0078 to 0.0182) is also significant when the population is estimated from the sample. <0.05.

ㆍ The result of co-dominant is determined by the risk allele (A2) from the result of allele analysis, and this allele is the same as the relative allele (A1) and gene expression. When sexually active, it is called co-dominant, and the independent variable for genotyping for risk is A1A1: A1A2: A2A2. The risk, 95% confidence interval, and significance level are interpreted as in the analysis of alleles.

ㆍ Dominant results are determined by the risk allele (A2) from the result of allele analysis, and when the risk allele acts dominantly in the relative allele (A1) and gene expression, the dominant (Dominant) ), And the independent variable of genetic tongue to determine the risk is A2A2 + A1A2: A1A1.

ㆍ Recessive results are determined by the risk allele (A2) from the result of allele analysis, and if this risk allele is enthusiastic in the expression of the relative allele (A1) and genes, it is recessive. The independent variable of genotype to determine the risk is A2A2: A1A2 + A1A1.

As shown in Table 2a, Table 2b, Table 3, and Table 4, the polymorphic markers of SEQ ID NOs: 1 to 10 of the present invention are 0.0078 to 95% confidence at 95% confidence, as a result of chi-square analysis between the expected and observed values of the allele frequency. The range of 0.0182 was significantly different from the control group, and the odds ratio ranged from 5.4 to 5.8.

The monobasic polymorphisms of SEQ ID NOS: 1 to 10 showed a significant frequency of appearance in the early menopausal patient group and normal subjects. Therefore, the monobasic polymorphism of the present invention can be effectively used for diagnosis or treatment associated with early menopause. Specifically, it may be used as a primer or probe for the diagnosis of premature menopause.

The present invention also includes allele-specific polynucleotides for early menopause diagnostics that hybridize with at least 10 consecutive polynucleotides or their complementary nucleotides derived from the DNA sequences of SEQ ID NOs: 1 to 10 including polymorphic sites. The allele-specific polynucleotide means to hybridize specifically to each allele. That is, it means that the single base polymorphism can specifically hybridize to one of the alleles represented, wherein hybridization may be performed at 48 ° C. under RA1 solution (Illumina's InfiniumTM Assay kit).

The invention also includes an allele specific primer for amplification for early menopause diagnosis, consisting of polynucleotides or complementary polynucleotides thereof. “Primer” herein refers to the synthesis of template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates (ATP, GTP, CTP, TTP) and DNA polymerase) in a suitable buffer and at a suitable temperature. It refers to a single stranded oligonucleotide that can act as a starting point. The appropriate length of the primer may vary depending on the purpose of use, but is usually 10 to 100, preferably 10 to 80 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. It is preferred that the primer 3'-terminal alignment with the single nucleotide polymorphism of SEQ ID NO: 1 to 10. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification in allelic form of DNA where the primer exhibits complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. The product is amplified from two primers by amplification, and only the amplified product is specifically stained and visualized. This means that certain allelic forms exist. If the allele-specific polynucleotides used herein undergo different denaturation processes, they may be used by other methods as well as Infinium ^™ Assay.

The present invention includes a premenopausal diagnostic probe and a premenopausal diagnostic microarray comprising the polynucleotide (a) to at least one selected from the group consisting of (j) or a complementary polynucleotide thereof. The present invention also includes a kit for early menopause diagnostic, comprising at least one polynucleotide selected from the group consisting of polynucleotides (a) to (j) or complementary polynucleotides thereof.

The present invention provides a method for obtaining a nucleic acid sample from a sample and determining a nucleotide sequence of one or more polymorphic sites of the polynucleotides of SEQ ID NOs: 1 to 10 or complementary nucleotides thereof, wherein the polymorphic sites are as defined above. Includes early menopause diagnostic methods.

Wherein determining the nucleotide sequence of the polymorphic site comprises hybridizing the nucleic acid sample to a polynucleotide selected from the group consisting of polynucleotides (a) to (j) or a microarray to which complementary nucleotides thereof are immobilized and obtained Detecting the hybridization result.

In another embodiment of the method of the present invention, as a result of determining the nucleotide sequence of the polymorphic site, the bases of the polymorphic sites of SEQ ID NO: 1 to 10 are C, C, T, A, T, G, T, A, C, respectively. And, if one or more of A (risk allele) is present, determining that the group is at high risk of premature menopause. The more the nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to the risk group can be determined.

The present invention includes obtaining a nucleic acid sample from a sample and detecting the presence of a haplotype in a BCKDHB (Branched Chain Keto acid DeHydrogenase E1, Beta polypeptide) gene comprising a DNA sequence of SEQ ID NOs: 1-10. Includes diagnostic methods for the presence or absence of early menopause.

That is, according to another embodiment of the present invention, obtaining a nucleic acid sample from a sample; And when the haplotype analyzed from the polymorphic site of the DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 10 is TCCAAGGGCT, a diagnosis method for premenopausal presence is provided. In addition, in the above method, when the polymorphic sites of the DNA sequences of SEQ ID NO: 1 and SEQ ID NO: 3 are represented by T and C as a tagging SNP, they may be determined to be normal.

In addition, according to another embodiment of the present invention, obtaining a nucleic acid sample from a sample; And when the haplotype analyzed from the polymorphic site of the DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 10 is CATTGCAATC, there is provided a diagnostic method for the presence of early menopause comprising the step of determining the risk group with a high risk of early menopause. In addition, in the above method, when the polymorphic sites of the DNA sequences of SEQ ID NO: 1 and SEQ ID NO: 3 appear as C and T as the tagging SNPs, it may be determined that the risk group has a high risk of premature menopause.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example

Subjects

The experiment was conducted with 16 patients who had been diagnosed as early menopause and 16 normal patients who had been diagnosed as early menopause by the Korean Tea Hospital. They all had karyotypes of 46 and XX. All normal people were selected from people with children. The clinical criteria for early menopause were defined as blood follicle stimulating hormone levels (FSH levels) of 40 mIU / mL or higher. And height (cm), weight (Kg), body mass index (BMI (Kg / m ² )), luteinizing hormone (LH (mIU / mL)), estradiol (E ₂ (pg / mL)), prolactin (PRL) (ng / mL)), testosterone (ng / mL), and thyrotropin (TSH (mIU / mL)) were investigated. In 16 early menopausal patients, 5 had undergone IVF and 1 had failed IVF. In the family history survey, there were two patients with amennorrhea among mothers and siblings.

2. Preparation of Genomic DNA

Blood was collected from 16 premenopausal patients and 16 normal women to prepare genomic DNA (gDNA). 5 ml of total venous blood was obtained from each volunteer's anterior venous vein and transferred to a Vacutainer tube containing 15% EDTA. To extract genomic DNA, the blood is transferred to a centrifuge tube and a low concentration of salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA) is added. 5 ml was added. And Nonidet P-40 was added. The resulting solution was mixed well and centrifuged at 2,200 rpm for 10 minutes at room temperature. The obtained mass was resuspended in a high concentration salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , 0.4M NaCl, and 2 mM EDTA). The genomic DNA was mixed with 50ul of 10% SDS and reacted overnight at 55 ° C.

After the reaction, each genomic DNA was transferred to a 1.5 ml centrifuge tube and 0.3 ml of 6 M NaCl was added. After mixing the genomic DNA well, it was centrifuged for 10 minutes at 12,000rpm. After centrifugation, the upper layer containing genomic DNA was collected and transferred to a new test tube, and the same amount of 100% ethanol was added at room temperature. Mix well until genomic DNA precipitates and wash genomic DNA with 70% ethanol when precipitated. At the end of this process, open the test tube lid and let the solution dry. Then, TE buffer solution (pH 8.0) was added to resuspend the genomic DNA.

Prepared genomic DNA using the ^{Quant-iT TM PicoGreen dsDNA reagent (} Molecular Probes, Invitrogen) for the ^TM Infinium Assay was adjusted to a concentration of 750ng / 15uL.

3. Genotyping of the Whole Genome-Infinium ^TM Assay

Description of the Infinium ^TM Assay Infinium ^TM Assay is to allele probe which can distinguish (allele A and allele B) for each single nucleotide polymorphisms as microarray using Sentrix ^R BeadChip (Illumina) planted in a bead . These probes consist of about 75 nucleotides, of which 25 nucleotides on the 5 'side are able to distinguish each single nucleotide polymorphism, while the remaining 50 nucleotides are designed to distinguish alleles for each single nucleotide polymorphism. have.

Whole - genome amplification amplification ( WGA )) Genomic DNA (750ng / 15uL) was dissolved at room temperature (22 ° C) and transferred to each well of 0.8mL micotiter plate. 15 uL of 0.1N NaOH was added and reacted at room temperature for 10 minutes. After the reaction, add 270uL of MP1 solution and 300uL of AMM solution in turn, and mix well. The reaction was centrifuged at 280g for 20 hours in a 37 ℃ oven.

At the end of the fragmentation and purification total genomic DNA amplification phase of the amplified genomic DNA was separated one minutes and centrifuged at 50g, were transferred to the three extra per 150uL well. As a result, four wells become identical genomic DNA. 50 μL of FRG solution was added to each well, followed by vortex for 1 minute at 1600 rpm. After centrifugation at 50g at room temperature, the reaction was carried out at 37 ℃ for 1 hour. Genomic DNA was centrifuged at 50g, 100uL of PA-1 solution was added and vortexed at 1600rpm, centrifuged at 50g at room temperature, and left at 37 ° C for 5 minutes. After centrifugation at 50g for 1 minute at room temperature, 300uL of 100% isopropanol was added and reacted at 4 ° C for 30 minutes. After centrifugation at 3000g for 20 minutes at 4 ℃ to confirm the genomic DNA clumps, the top layer was removed by inverting the microtiter plate. After drying the genomic DNA for 1 hour at room temperature, 42uL of RA1 solution was added, and reacted for 1 hour at 48 ℃ oven.

Sentrix ^R Preparation of BeadChips and (Sentrix ^R ) BeadChips were prepared while resuspending the activated amplified genomic DNA in RA1 solution. The BeadChip was taken out and shaken up and down about 20 times in 100% ethanol and shaken with a reaction time interval of 5 minutes and 10 minutes. After the reaction in 100% ethanol, the BeadChip was shaken by transferring to PB1 solution, and shaken at the interval of 2.5 minutes and 5 minutes in the same manner as in 100% ethanol. After centrifugation at 280g for 1 minute at room temperature, it was assembled in the recommended order (from Illumina) to prepare a BeadChip Chamber (called Flow through chamber). 150 uL of 100% formamide was passed through a BeadChip Chamber and 150 uL of RA1 solution was passed through twice. The BeadChip Chamber was placed in a Hybridization chamber containing PB2 solution until genomic DNA was added.

After resuspension of hybridized amplified genomic DNA (48 ° C., 1 hour reaction), vortex for 1 minute at 1800 rpm, centrifuge at 280 g, and denature amplified genomic DNA for 20 minutes in a 95 ° C. heat block. , Centrifuged at 280 g. Genomic DNA divided into four wells was pooled into one well and centrifuged at 280g. After removing the BeadChip Chamber from the Hybridization Chamber and adding genomic DNA, the BeadChip Chamber is placed in the Hybridization Chamber again and shaken at 48 ° C. for 18 hours to react.

Allele-specific primer extension (Allele - Specific Primer Extension ; After 18 hours of hybridization with ASPE ) , the BeadChip chamber was taken out and flowed four times with 450 uL of RA1 solution. 450uL of the XB1 solution, 450uL of the XB2 solution, and 200uL of the EMM solution were sequentially reacted for 10 minutes, 5 minutes, and 15 minutes, respectively, and 450uL of 95% formamide / 1 mM EDTA was flowed. 450 uL of XB3 solution was flowed and genomic DNA was stained with LMM and ASM solution according to Illumina's protocol. After disassembling the BeadChip chamber, the BeadChip was washed with PB1 solution, centrifuged at 280g for 1 minute, dried, and scanned using an Illumina BeadArray reader to store image files for each BeadChip.

Genotyping Analysis of genotypes from image files was performed using Illumina's GenCall software (version 6.2.0.4) for genotyping for each monobasic polymorph. GenCall exhibits a typical genotype pattern with a percentage of the intensity of color development between probes recognizing two alleles and is reported as a GenCall score.

4. Statistical Analysis

Genotypes for each of the single nucleotide polymorphisms were analyzed using Haploview software (version 5.0) and HapAnlayzer (version 1.1). First, the minimum allele frequency (MAF)> 0.1 was used as the basis for each monobasic polymorphism. The significance level (p) was determined by using the Hardy-Weinberg Equilibrium (HWE) test with HapAnalyzer. single nucleotide polymorphs were selected. From the results selected, alleles and genotypes were analyzed for association with disease to determine the odds ratio (OR) for each and significant differences (<0.05) between the two groups from the significance level. ) Was examined. In addition, we conducted a linkage disequilibrium (LD) between single nucleotide polymorphisms that were closely related at the genetic level using Haploview (Fig. 1, Table 5). One association was also tested (FIG. 2).

The chi-square test was used for the Heidi-Wineberg test and the haplotype association, and the logistic regression was used for the analysis of risk and 95% confidence interval (CI). In addition, Lewontin's D 'and R ² measurements were used for the association of each polybasic polymorphism by the association equilibrium assay.

5. Observation on the analysis of monobasic polymorphs

From the results of each monobasic polymorphism, it can be seen that certain monobasic polymorphisms are closely related to each other and form a block unit, and these block units are called associative non-equilibrium blocks. It can be expected to move together at chromosome crossing over. Also, by analyzing the observed genotypes, the haplotypes of the associative non-equilibrium blocks can be determined (Fig. 1 and Table 5).

Table 5 shows the numerical value of the compactness of the associative non-equilibrium blocks of the single base polymorphisms shown in FIG. 1, and the lower part of the diagonal line represents the D 'value. When the value of D 'is 0.8 or more, it is determined that each of the monobasic polymorphisms has an associative non-equilibrium relationship and is shown in red as shown in FIG. The range of such D 'value is 0-1. In addition, the R ² value represents the correlation between each of the single nucleotide polymorphisms. If the R ² value is 0.5 or more, the single nucleotide polymorphisms corresponding to the value are closely packed. This means that in the aforementioned FIG. 1 and Table 5, the ten monobasic polymorphs are closely related to each other to form a genetic unit. In addition, these formed units are bundled together and transferred when passed to the next generation. Thus, non-equilibrium blocks appear to be patient-specific and cause disease, and if so affected, the block itself is passed on to the next generation, thus predicting the frequency of disease in the offspring of the next generation.

Genetic units formed from associative non-equilibrium blocks are determined from the genotyping of each monobasic polymorphism, which is called haplotype (FIG. 2).

FIG. 2 is a haplotype formed in comparison to genotyping results from the associative non equilibrium block formed from FIG. There are three haplotypes, the two most common being 'TCCAAGGGCT' and 'CATTGCAATC'. Since haplotypes are passed down to the next generation, it is necessary to analyze whether these haplotypes are associated with disease. Overall, disease-related analyzes of associative non-equilibrium blocks and haplotypes can predict not only the diagnosis of the disease in the current generation, but also the transmission of the disease to the next generation. In addition, small inverted triangles are indicated in the first and third nucleotide sequences of each haplotype, indicating that the single base polymorphism present at the site represents the corresponding haplotype. For example, even if all the top haplotypes are not analyzed, if the single-base polymorphs in the first and third positions are represented by T and C, the result can be judged as the highest haploid type. It means. Such single nucleotide polymorphisms that can represent haplotypes are called tagging SNPs.

Table 6 shows the results of the chi-square test showing whether the three types of haplotypes determined from associative non-equilibrium blocks are significantly different in the patient group and the normal group. From this result, it can be seen that the haplotype of the highest is significantly higher (p value <0.0034) in the normal group. The haplotype of the second type shows that the overall frequency is low but is significantly higher (p value <0.0079) in the patient group than in the normal group.

In view of the results of such an analysis, each allele may exist in the form of a homozygote or heterozygote in each individual. Mendel's genetic law and Hardy-Weinberg law show that the composition of the alleles that make up a group is maintained at a constant frequency and, if statistically significant, can give a biological function meaning. It can be seen that the monobasic polymorphism of the present invention appears in a statistically significant level in premenopausal patients, and can also be used for diagnosis and further prediction of early menopause from these monobasic polymorphisms to genetic units.

1 is a diagram showing a haploid shape of an associative non-equilibrium block;

Figure 2 shows the haplotypes formed in comparison to genotyping results from associative non-equilibrium blocks.

<110> SUNGKWANG MEDICAL FOUNDATION <120> Methods for diagnosing premature ovarian failure using          polynucleotides comprising single nucleotide polymorphism <130> PN0155 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 401 <212> DNA <213> Homo sapiens <400> 1 actttatacc attgtttcaa cttcaacctt tatttttgta tatatttttt cagctactta 60 aactgttcat gaataggcgt actttctgta tttaaaaatg gcccctcaag caccgttaat 120 ttacattcca gttatttaca tgataattca tgacattctg aaacttgcct gtatattatc 180 tgaaaaatgg atttcttgag saaaagatct gtttattgta tgtaaggaaa aattttacct 240 gaaaacaaac aaacaaaccc taaaactcag caccactacc atttccagaa gcttttttaa 300 caagtgaata ttttttacat aatggataat aaatggtatt tattcatcat gattttctaa 360 gtaatgctta tcagcccctt cagtggtgtt attctaacaa a 401 <210> 2 <211> 401 <212> DNA <213> Homo sapiens <400> 2 aaaacaactc caatatgcta aaagttaaat atggtattta agaaaatagt catgtatgca 60 attgagaaag tctataattt attttacaga aaagctggaa gattatgact agatatggaa 120 atattttttc tgaatttttt tttatatttc ctccgactta cctctttttg aaaagagagt 180 ttttattaag tgaaccatca ygatattggc tgaaaagttc tacattctat tattgtattg 240 taacacacat gtattgatga ttttcattaa gagtttcaga ttaactttga aaaatattcc 300 acatggtaat cttataaatt ctgtttaatt acatctgtaa atattatgtg tgtgatagta 360 ttcaataaag taaaatcaaa ttgtctttgg gttgtatgtg t 401 <210> 3 <211> 1548 <212> DNA <213> Homo sapiens <400> 3 atggtgcatt aaatttcatc attttcaatt cctagtcccc cccaaatgag catgcttaca 60 attgacataa caaataacca acccccaagg attttcttta ggtttcttca aatgctaatt 120 atgtagcaaa yctaattaag ctcaagtgaa tcacttcagc agtaggtact ttggttactt 180 cataaatgcc aaaagggatg agaagtgtct attctccctc caaagcagtc atgctgtgtg 240 ccaaactgat aactgttaaa cacagctgca gagcaaatgt gcagttttgg tcctgtaaat 300 acagtgttca cacatcaacc tgcgctgtga gtgatgtaga attcagaaca accgactgca 360 gattggctca tgttacatga gtattataga tgagtgctgt ttcacccagc acagcttgag 420 cagattttga tgggccatga aagttccctc tacctaagaa tatatactga aactctaaaa 480 accccaaata ttgataaatt tattggatca acatctggtt gaagtggttt gcttttaagc 540 tccatagctc acaagaataa aagtataaag gtcattttgt gtgtctgaga gcacatttat 600 ggaatggtta acacaaaaat ctattattta atgtcatccc tgaagaaata tatgctgtct 660 ttattataaa tgtttttcct ttaattccag taggtaaaag tgcaaaacaa acctataagc 720 aaatctaagc taaaataaac tctttttatt ttagaaactc aatttccatg gaaggaaatc 780 cttttcagag gggctctctc aactgggtgg gttgccggat gcttaaggcc atgactgcat 840 tccctccaga ggctgttaga tcactatggc aactaacttt tccctatcaa taagcaagag 900 ctacaaacgc taatattctg aaaaagccat ttccttttct gtggcattaa caatataacc 960 tagtttcaag atagaaatat acctcttttt gtgcaaaacg attatctttg ctggtaatta 1020 ctagaattca gctttgcaaa taaatgcaaa aatagcaaaa tcagccactg tacttcagtt 1080 tcaaaacaag gtaaaatttg tgacatagta atctaaggat ggtacagcta aagtgggttg 1140 ggtgtgtagg tctgggcaga aaaaaaattg ggtattttag gagcaaatgt catatgagaa 1200 ggaaaactga caagaaaaca acacatgctt ccacgtagct cttggagttt cttaaaagaa 1260 gcacacattt caacagttat atttcatacc tgtagttgtt tctagatatc taattcttac 1320 tgtgtagggt aacaaataaa ttgacatttt ccaaattaaa tacatatcct tcctatttag 1380 ggaggtattt gccagctttg tcttcactgg ttcgagtatc aacagctaaa tctcttaagt 1440 ggtgtctcat tttaggtatg caaaaacatc tccaagtatc acatacacag atttacaagc 1500 ccaaatggat cagaggtaca atgactgaca tggagcaggg atcagtaa 1548 <210> 4 <211> 401 <212> DNA <213> Homo sapiens <400> 4 aggcgtttca ctctgatttt cagtgagcta gatttactac ccatacacaa aagtcaagaa 60 ttctagcatt tctgatcttt aaaaagtaga gaatgtgcta ttatacagag ttgattttgc 120 ttttgaaaaa gcatagttct tcaaaagata ttactatcat ttttggaaaa actagatatt 180 aaaaggtaaa ggatctaatc rtatctactc attttgaaac atttcaaaat cagtgtgtca 240 gtgctagtaa cttctatatt aaaactctga aaagctaact taataatttg taatgtattc 300 attttgttag aataacacca ctgaaggggc tgataagcat tacttagaaa atcatgatga 360 ataaatacca tttattatcc attatgtaaa aaatattcac t 401 <210> 5 <211> 401 <212> DNA <213> Homo sapiens <400> 5 ctagcaactg gaaatctcca cgaacaactt taggactttt aatacacacg gtttaaagag 60 ccacctaaga gattccacta ctaataagct gaaaagagat gacagactgg gtggattatt 120 tcttgtttgc aacaagtaat tttaaagggc tttaaattgt caatttggga ttaagtcggt 180 tttattactt tagctgacac wgggctgttt ttccatcttc cctgaatact gaactcttca 240 taaacaagct atccatggag aattaagggc aaaatatttt actaaacgtc tttgatttta 300 tacctgttat aaaatgactg ttctcttgaa ccagtaactt ggaaagctgg cctaccacaa 360 gaagttctgt tggtatttgt taaaatcact cctttcagca t 401 <210> 6 <211> 1005 <212> DNA <213> Homo sapiens <400> 6 acatttttgt tgttgttgtt gttctgagat ggagtctcac tctgtcgccc aggctagact 60 gcagtggtgc aatctcagct cactgcaacc tcccccctac ccctgagttc aagcgattct 120 cctgcctcag cctgctgagt agttgggatt acaggcgccc accaccatgc ccagctaatt 180 tttgtgtttt tattggaggt ggggtgtcac tatgttggct cagctgattt caaattcctg 240 acctcaagtg atccacctgc cttagcctcc caaagtgctg ggattacagg catgagccac 300 tgcacctggc tatatttaca tttaatagaa acatatctag catatgtata tgtgattttt 360 tttttttttt tgagaccgag tctcactctg tcaccaggct ggagtgcagt ggtgcgatct 420 tggctcatgg caacctctgc ctcccaggtt caagcgattg tcctgcctca gtctcctgag 480 tagctgggac tacaggtgtg caccaacatg cccagctaat tttttatatt tttagtagag 540 acggggtttc accatgttgg ccaggatggt ctcgatctct tgaccttgtg ttccacccgc 600 cttggcctcc caaagtgctg gaattacagg catgagccac cgcgcctggc ctgtatatgt 660 gatttctaaa aaatagatgc atgcatatgt taacattgaa tagtcaatca ctagatgaag 720 atgctctcta ccatggttta gattgcaagt gtactttata ccattgtttc aacttcaacc 780 tttatttttg tatatatttt ttcarctact taaactgttc atgaataggc gtactttctg 840 tatttaaaaa tggcccctca agcaccgtta atttacattc cagttattta catgataatt 900 catgacattc tgaaacttgc ctgtatatta tctgaaaaat ggatttcttg agcaaaagat 960 ctgtttattg tatgtaagga aaaattttac ctgaaaacaa acaaa 1005 <210> 7 <211> 401 <212> DNA <213> Homo sapiens <400> 7 tgctaccata atccctacat tttctgtctg ttctagtcta agaatattgt tatagatgga 60 agttaggacc attagcccac agatgcatgt attctctcag acataccggt gtagatgcta 120 tatttctgat gtcttcttaa tgtctgtgaa agcaactggc atctacaata aatcacactt 180 agagctggtt agaggactcc ytcacttttg ttgtccatgt ggttcccttc cgtggaccag 240 ccgtaataaa gagccaaggt agtgatggtg gccacgtgcc tcgttgctat ttttaaagta 300 atattcagat gtggttttaa atttagatta tgtattcctt ttgaaacata agaaaaacat 360 ttaaacctat gctgaaaata cgataaaaga aaaacaactc c 401 <210> 8 <211> 401 <212> DNA <213> Homo sapiens <400> 8 gctagaattc ttgacttttg tgtatgggta gtaaatctag ctcactgaaa atcagagtga 60 aacgccttta catttgtgcg gatagagaag ttaactctcc ctcatatgtc actgtgctac 120 cataatccct acattttctg tctgttctag tctaagaata ttgttataga tggaagttag 180 gaccattagc ccacagatgc rtgtattctc tcagacatac cggtgtagat gctatatttc 240 tgatgtcttc ttaatgtctg tgaaagcaac tggcatctac aataaatcac acttagagct 300 ggttagagga ctccttcact tttgttgtcc atgtggttcc cttccgtgga ccagccgtaa 360 taaagagcca aggtagtgat ggtggccacg tgcctcgttg c 401 <210> 9 <211> 401 <212> DNA <213> Homo sapiens <400> 9 acattttctc tgctgtccca gaaagttggc tttcaatatc ttaaccctgt tttatctttt 60 ttaaccttaa aaaatatacc ccaaaaaaga aatagagaaa tatgaatcat gctaagtggt 120 gaaagcagtt gtacctcaaa cttaggtgat ctcagtttaa acaacccaag gcactggtaa 180 tttgatacag gtttcactta yaatatggaa caaggctttc catagagagg aagctcaata 240 caggatattg catagttgaa tatctgcatg aaagagttac tgaaaggtat attttattac 300 tcagattact caaaattata tgggaaaatt atcaatgaaa aaaaattttt caagtgtcta 360 gttttttttc taatcttctt tatcagtaga taaaagccat a 401 <210> 10 <211> 701 <212> DNA <213> Homo sapiens <400> 10 ccctcaatag gctaggcatt gaaggaacat acctcaaaac aataaaagcc atctatgaca 60 aacccacagc taacatcata ctgaatgagc aaaagctgga accattccac ttgagaaccg 120 gaacaagaca aggataccca ctctcaccaa tcctattcaa gatagtactg gaagtcctgg 180 ccagagcaat caggcaagag aaagaaataa aaggcattca aataagaaat gaagaaatca 240 aactggctct cttcattaaa aatatgattt tatacttaga aaaccctaca gactctgcca 300 aaaggctact agaactgata aacaatttta gcaaggtttc aggatacaaa atcaatgtac 360 aaaaatcagt agcatttcta tatacagtaa tgtctaattg aaatatatac taaattattt 420 ttaaaacaac ttttagattt tttcttacaa atttttgcaa tgatgttgtg aggatgcttt 480 ccttttgcaa aaggcttatt mcataccttt tccttttgca aaatgtttag ttctgtaatc 540 atgacataat caacatgaga catgttagag gattggatgt tcaaaatgct gactttcctg 600 gcaaaaccta tgttgtcaag taggtgataa aagtgttttc taataaaaca agcacagtgc 660 ttatttgtta tatgtcagtg caaaccaaat gagcttgagt t 701 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> haplotype <400> 11 tccaagggct 10 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> haplotype <400> 12 cattgcaatc 10  

Claims (11)

A method of analyzing a monobasic polymorphism of a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NOs: 1 to 10 using primers or probes whose template is a polynucleotide sequence selected from the group consisting of polynucleotides of SEQ ID NOs: 1 to 10 , The monobasic polymorph has 10 or more contiguous DNA sequences (hereinafter referred to as "polynucleotide (a)") in which the 201 base (polymorphic site) of the DNA sequence of SEQ ID NO: 1 is C and the 201 base is included. , 201 base (polymorphic site) of the DNA sequence of SEQ ID NO: 2 is C, 10 or more consecutive DNA sequences comprising the 201 base (hereinafter referred to as "polynucleotide (b)"), SEQ ID NO: 3 Of the DNA sequence of SEQ ID NO: 4, wherein the 131th base (polymorphic site) of the DNA sequence of T is T, and the 10 or more consecutive DNA sequences comprising the 131th base (hereinafter referred to as "polynucleotide (c)") At least 10 contiguous DNA sequences (hereinafter referred to as "polynucleotide (d)") comprising the 201 base (polymorphic site) A and the 201 base (the 201 base of the DNA sequence of SEQ ID NO: 5) Polymorphic site) T and at least 10 contiguous DNA comprising said 201 base Column (hereinafter referred to as "polynucleotide (e)"), the 805th base (polymorphic site) of the DNA sequence of SEQ ID NO: 6 is G and at least 10 consecutive DNA sequences comprising the 805th base (hereinafter, "Polynucleotide (f)"), the 201 base (polymorphic site) of the DNA sequence of SEQ ID NO: 7 is T and at least 10 contiguous DNA sequences comprising said 201 base (hereinafter "polynucleotide ( g) ", the 20th base (polymorphic site) of the DNA sequence of SEQ ID NO: 8 is A and at least 10 contiguous DNA sequences comprising the 201th base (hereinafter referred to as" polynucleotide (h) "). At least 20 contiguous DNA sequences (hereinafter referred to as “polynucleotides (i)”) and sequences comprising the 201 base of the DNA sequence of SEQ ID NO. The 501th base (polymorphic site) of the DNA sequence of No. 10 is A, and the 501th base comprises 10 Or more contiguous DNA sequence (the "polynucleotide (j)" hereinafter) in, characterized in that consisting of selected from the group consisting of the DNA sequence, A method for analyzing monobasic polymorphisms of polynucleotides selected from the group consisting of polynucleotides of SEQ ID NOs: 1-10. The method of claim 1, wherein the DNA sequence is 10 to 100 nucleotides in length. Obtaining a nucleic acid sample from the sample to provide information necessary for early menopause diagnosis; And determining a nucleotide sequence of at least one polymorphic site of the polynucleotides of SEQ ID NOs: 1 to 10, wherein the polymorphic site is the 201 base in the case of the DNA sequence of SEQ ID NO: 1, wherein the base is C, and In the case of DNA sequence, the base is C as the 201 base, in the case of the DNA sequence of SEQ ID NO: 3, the base is T as the 131th base, in the case of the DNA sequence of SEQ ID NO: 4, the base is A as the 201 base, In the case of the DNA sequence of SEQ ID NO: 5, the base is T as the 201 base; in the case of the DNA sequence of SEQ ID NO: 6, the base is G as the 805 base; in the case of the DNA sequence of SEQ ID NO: 7, the base is the 201 base Is T, the base is A as the 201 base for the DNA sequence of SEQ ID NO: 8, the base is the C as the 201 base for the DNA sequence of SEQ ID NO: 9, and the DNA of SEQ ID NO: 10 Method of analyzing a single nucleotide polymorphism in a sample containing the said base is A) a five hundred and first base when the column. The method of claim 3, wherein the determining of the nucleotide sequence of the polymorphic site comprises hybridizing the nucleic acid sample to a microarray to which a polynucleotide selected from the group consisting of polynucleotides (a) to (j) is immobilized. Polynucleotides (a) to (j) are as defined in claim 1); And detecting the obtained hybridization result. delete delete delete delete delete Obtaining a nucleic acid sample from the sample to provide information necessary for early menopause diagnosis; And a polymorphic site of the DNA sequence of SEQ ID NOs: 1 to 10, wherein the polymorphic site is the 201 base for the DNA sequence of SEQ ID NO: 1, the 201 base for the DNA sequence of SEQ ID NO: 2, and the DNA sequence of SEQ ID NO: 3 In case of 131 base, in case of DNA sequence of SEQ ID NO: 4, in base 201, in case of DNA sequence of SEQ ID NO. As the haplotype determined from the 20th base, the 201 base for the DNA sequence of SEQ ID NO: 8, the 201 base for the DNA sequence of SEQ ID NO: 9, and the 501th base for the DNA sequence of SEQ ID NO: 10). And analyzing whether the haplotype is TCCAAGGGCT or not. Obtaining a nucleic acid sample from the sample to provide information necessary for early menopause diagnosis; And a polymorphic site of the DNA sequence of SEQ ID NOs: 1 to 10, wherein the polymorphic site is the 201 base for the DNA sequence of SEQ ID NO: 1, the 201 base for the DNA sequence of SEQ ID NO: 2, and the DNA sequence of SEQ ID NO: 3 In case of 131 base, in case of DNA sequence of SEQ ID NO: 4, base 201, in case of DNA sequence of SEQ ID NO: 5, base 201, in case of DNA sequence of SEQ ID NO: 6, base 805, in case of DNA sequence of SEQ ID NO: 201 As the haplotype determined from the 20th base, the 201 base for the DNA sequence of SEQ ID NO: 8, the 201 base for the DNA sequence of SEQ ID NO: 9, and the 501th base for the DNA sequence of SEQ ID NO: 10). And analyzing whether the haplotype is CATTGCAATC.

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