KR101100323B1 - Polynucleotides derived from PCMT1 gene comprising single nucleotide polymorphism, microarrays and diagnostic kits comprising the same, and analytic methods using the same - Google Patents

Abstract

The present invention provides a polynucleotide comprising a single-nucleotide polymorphism (SNP) derived from a Protein L-isoaspartate O-methyltransferase (PCMT1) gene or a complementary polynucleotide thereof, and the polynucleotide or a complementary polynucleotide thereof. Polynucleotides can be used as markers for clinical diagnosis of Premature Ovarian Failure (POF).

PCMT1, early menopause

Description

Polynucleotides derived from PCMT1 gene containing single nucleotide polymorphism, microarrays and diagnostic kits comprising the same, and analytic methods using the same}

The present invention provides a polynucleotide comprising a monobasic polymorphism derived from the PCMT1 gene or a complementary polynucleotide thereof; An amplification primer or early menopause diagnostic probe for diagnosing early menopause comprising the polynucleotide or its complementary polynucleotide; A microarray comprising the probe; And an analysis method for providing information necessary for early menopause diagnosis using the polynucleotide or its complementary polynucleotide.

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 genes. 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 represented by transposable elements, short tandem repeats (STRs), microsatellites, sequence tagged sites (STS), variable number tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs). Known. 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 meaning in the genetics represented by 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.

In another aspect, monobasic polymorphs can be used as markers on the genome. This means that monobasic polymorphisms provide information about the different kinds of diversity that occur in genes. For example, if a single nucleotide polymorphism selected by associative equilibrium analysis is associated with an equilibrium equilibrium, alleles of that single nucleotide polymorphism are due to changes in the genetics occurring within those monobase polymorphisms. By analyzing, it is possible to know whether the mutation on the gene. Therefore, the monobasic polymorphism has a meaning as an important clue that can identify what disease is caused by changes such as mutations in the gene. Such associative equilibrium can predict important parts of recombination in genomic DNA, thus providing clues for predicting inheritance and / or genetic disease to the next generation.

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 stops, which is called menopause. Normal menopause occurs between about 45 and 56 years of age, but early menopause, or early ovarian failure, occurs in about 1% of women. Early menopause causes primary amenorrhea and secondary amenorrhea. Primary amenorrhea refers to the absence of menstruation, and secondary amenorrhea refers to loss of ovarian function before age 40. Early menopause predates menopause before the age of 40, and is characterized by increased follicle stimulating hormone secretion and decreased estrogen secretion in patients. Causes include genetic factors, autoimmune diseases, radiation therapy, chemotherapy, ovarian destruction by surgery, and other causes. Premature menopause may cause symptoms such as hot flashes, mood swings, and decreased libido in the short term, and in the long term, may lead to increased risk of heart disease and osteoporosis. Entails. This is a social problem because this infertility can lead to family discord, leaving the suffering of the individual as a disease. Therefore, if a single base polymorphism is identified that may be specific to early menopausal patients, it can be used as a marker for predicting early menopause. It may be followed. In addition, we can predict how early menopause affects later generations.

Currently, monobasic polymorphisms associated with early menopause include several monobasic regions in the Follicle Stimulating Hormone Receptor, the Follicle Stimulating Hormone beta subunit, and the Inhibin alpha subunit. Has been confirmed. However, they did not show consistent results in other studies. This may be because monobasic polymorphisms have population specificity and early menopause is a polygenic disease affected by several genes. Therefore, there is a need in the art to find new monobasic polymorphic sites associated with premature menopause in Korean women.

The present inventors conducted a study to find a monobasic polymorphism specific to early menopause in women, especially Korean women. As a result, we found that the protein L-isoaspartate O-methyltransferase (PCMT1) gene is specific to patients with early menopause. The discovery of a base polymorphic site led to the completion of the present invention.

Accordingly, an object of the present invention is to provide a polynucleotide or a complementary polynucleotide thereof comprising a monobasic polymorphism derived from the PCMT1 gene as 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 comprising the polynucleotide or its complementary polynucleotide.

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

It is also an object of the present invention to provide an early menopause diagnostic method using the polynucleotide or its complementary polynucleotide.

According to one aspect of the invention, in the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the 201 base (polymorphic site) is A, the polynucleotide consisting of 10 or more consecutive DNA sequences comprising said 201 base ( a); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the 301th base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, wherein the 301th base (polymorphic site) is A, and the polynucleotide (d) is composed of 10 or more consecutive DNA sequences comprising the 301th base. Polynucleotides selected above or complementary polynucleotides thereof are provided.

In addition, according to another aspect of the present invention, there is provided a primer for amplification for early menopause diagnosis, consisting of the polynucleotide or its complementary polynucleotide.

Further, according to another aspect of the present invention, there is provided a premenopausal diagnostic probe and a microarray comprising the polynucleotide or a complementary polynucleotide thereof.

In addition, according to another aspect of the present invention, there is provided a kit for early menopause diagnostic comprising the polynucleotide or a complementary polynucleotide thereof.

In addition, according to another aspect of the present invention, to provide information necessary for the diagnosis of early menopause, (i) obtaining a nucleic acid sample from a sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from the polynucleotides of SEQ ID NOS: 1 to 4 or its complementary polynucleotides from the nucleic acid sample, provided that the polymorphic site is For the DNA sequence, the 201 base, the 301 base for the DNA sequence of SEQ ID NO: 2, the 401 base for the DNA sequence of SEQ ID NO: 3, and the 301 base for the DNA sequence of SEQ ID NO: 4). A method for detecting a monobasic polymorph in a sample is provided.

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

In addition, a primer or probe consisting of the polynucleotide or its complementary polynucleotide, and the polynucleotide or the complementary polynucleotide kit thereof may be usefully used for predictive diagnosis of early menopause.

In addition, the analysis method of the present invention can be usefully used to provide information necessary for the diagnosis of early menopause.

The present invention provides polynucleotides or complementary polynucleotides selected from the group consisting of polynucleotides (a) to (d) derived from the sequences of SEQ ID NOs 1 to 4 newly found to be associated with premature menopause. That is, the present invention provides a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, wherein the 201 base (polymorphic site) is A, and is composed of 10 or more contiguous DNA sequences comprising the 201 base (a); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of 10 or more consecutive DNA sequences comprising a 301 th base (polymorphic site) A and comprising the 301 th base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, wherein the 301th base (polymorphic site) is A, and the polynucleotide (d) is composed of 10 or more consecutive DNA sequences comprising the 301th base. Provided above is a polynucleotide selected or complementary polynucleotides thereof.

Figure 1 shows the location of the monobasic polymorphs present in the PCMT1 (Protein L-isoaspartate O-methyltransferase) gene. The genes of SEQ ID NOS: 1 to 4 from which the polynucleotides (a) to (d) are derived are statistically evenly distributed in a sample group in which the patient group and the normal group are combined, which is a single of the polynucleotides (a) to (d). Base polymorphisms appear at high frequency and also mean that they can be used as stable genetic markers.

The DNA sequences of SEQ ID NOS: 1 to 4 are single nucleotide polymorphisms showing statistically significant difference (significance level p value <0.05) in the premenopausal patient group and the normal group, and they exist in the PCMT1 gene. The PCMT1 gene, located on chromosome 6, is an enzyme that acts as a protein repairer.It is a peptide or peptide that is formed by deparamidation of asparagine or isomerization of aspartate that occurs with age. Repairs naturally occurring isoaspartyl residues in proteins. The gene of SEQ ID NO: 1 (rs4816) is a monobasic polymorphism causing an amino acid change to change isoleucine to valine. As a result, two kinds of PCMT1 enzymes are generated. PCMT1 enzymes containing isoleucine are more stable, and PCMT1 enzymes containing valine have higher affinity for substrates. The difference in affinity between these two enzymes is about 30%, which can lead to significant differences in the accumulation of isoaspartyl residues. According to the allele frequency of the gene of SEQ ID NO: 1, valine was significantly higher in the normal group, and isoleucine was significantly higher in the patient group. PCMT1 is highly expressed in the mammalian pituitary gland, and the anterior pituitary hormones are effective substrates of the PCMT1 enzyme, which in particular exhibits high activity against follicle stimulating hormone (FSH) and luteinizing hormone (LH). This enzyme is also expressed in the human pituitary gland. Several aspartates are present in the receptor binding portion of human follicle stimulating hormone beta subunits. If the aspartate in this region is changed to isoaspartate by isomerization and inefficiently repaired by PCMT1, which contains isoleucine, which is significantly higher in the patient group, the structure of the receptor-binding portion of the follicle stimulating hormone may be altered. It is estimated. This may reduce the affinity of the follicle stimulating hormone for the receptor and may negatively affect the female reproductive cycle by interfering with follicle formation and ovulation.

The present inventors conducted a variety of studies to select PCMT1 as a candidate gene associated with early menopause, and to develop diagnostic indicators at the molecular level in early menopause diagnosis. In the present invention, the analysis was divided into two stages, and in the first stage, the normal group and the patient group participated in 24 persons each and were used for data analysis. In the second stage, 194 people and 108 people participated in the data analysis. In the second stage, both normal and patient groups of the first stage were included. As a result of genotyping and statistical analysis of the normal and the patient group, it was found that only a few of the single nucleotide polymorphisms showed a statistically significant difference between the normal and the patient group. In particular, the monobasic polymorphisms of SEQ ID NOS: 1 to 4 showed a risk allele at each polymorphic region as a result of Hardy-Weinberg Equilibrium and logistic regression analysis.

The present invention includes a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (d) or a complementary polynucleotide thereof, and the length of the polynucleotide or the complementary polynucleotide 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 (d) 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.

The present invention also includes an allele-specific primer for amplification for early menopause diagnosis, consisting of one or more polynucleotides selected from the group consisting of the polynucleotides (a) to (d) 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'-end is aligned with the monobasic polymorphism of SEQ ID NO: 1 to 4. 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, Infinium ^™ Assay I can be used by other methods as well.

The present invention includes a premenopausal diagnostic probe and a premenopausal diagnostic microarray comprising the polynucleotide (a) to at least one polynucleotide selected from the group consisting of (d) 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 (d) or complementary polynucleotides thereof.

In order to provide information necessary for the early menopause diagnosis, the present invention comprises the steps of: (i) obtaining a nucleic acid sample from the sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from the polynucleotides of SEQ ID NOS: 1 to 4 or its complementary polynucleotides from the nucleic acid sample, provided that the polymorphic site is For the DNA sequence, the 201 base, the 301 base for the DNA sequence of SEQ ID NO: 2, the 401 base for the DNA sequence of SEQ ID NO: 3, and the 301 base for the DNA sequence of SEQ ID NO: 4). It includes a method for detecting a monobasic polymorphism in the sample.

The sample includes blood, tissue, cells, and the like, and a nucleic acid sample can be obtained according to a conventional method. For example, in the case of blood, a nucleic acid sample can be obtained as follows. Transfer blood to a centrifuge tube and add low concentrations of salt component buffer (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA). After adding Nonidet P-40, the resulting solution was mixed well and centrifuged at about 2,200 rpm for 10 minutes at room temperature. The resulting mass is resuspended in a high concentration of salt component buffer (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , 0.4M NaCl, and 2 mM EDTA). Genomic DNA is mixed with 50ul of 10% SDS and reacted at about 55 ° C overnight. After the reaction, each genomic DNA is transferred to a 1.5 ml centrifuge tube and 0.3 ml of 6 M NaCl is added. After mixing the genomic DNA well, centrifuge for 10 minutes at about 12,000rpm. After centrifugation, the upper layer containing genomic DNA is collected, transferred to a new test tube, and the same amount of 100% ethanol is added at room temperature. Mix the genomic DNA well until it is settled, and wash the genomic DNA with 70% ethanol when settling. At the end of this procedure, open the test tube lid and allow the solution to dry. The nucleic acid sample can be obtained by resuspending genomic DNA by adding TE buffer solution (pH 8.0).

Here, the step of analyzing the nucleotide sequence of the polymorphic site is a sequence comprising a polymorphic site as a polynucleotide or a complementary polynucleotide sequence thereof selected from the group consisting of polynucleotides of SEQ ID NO: 1 to 4 (wherein the polymorphic site is For the DNA sequence of SEQ ID NO: 1, the 201 base, the 301 base for the DNA sequence of SEQ ID NO: 2, the 401 base for the DNA sequence of SEQ ID NO: 3, and the 301 base for the DNA sequence of SEQ ID NO: 4 It can be carried out using a primer or a probe as a template). The primer or probe is as described above.

In addition, the step of analyzing the nucleotide sequence of the polymorphic site is a step of hybridizing the nucleic acid sample in a microarray fixed with a polynucleotide selected from the group consisting of polynucleotides (a) to (d) or a complementary polynucleotide thereof ( Wherein the polynucleotides (a) to (d) are as defined above); And analyzing the obtained hybridization results, wherein the lengths of the DNA sequences of the polynucleotides or complementary nucleotides immobilized on the microarrays may be 10 to 100 nucleotides, respectively.

Results obtained according to the method for detecting a monobasic polymorphism in a sample of the present invention may provide information necessary for diagnosing early menopause. For example, the bases of the polymorphic sites of SEQ ID NOs: 1 to 4 are A, A, G, If more than one A (risk allele) is present, it can be determined that they belong to a risk group that is more likely to develop early 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.

In addition, analyzing the nucleotide sequence of the polymorphic site may comprise genotype (genotype) analysis of the polymorphic site of the DNA sequence selected from the group consisting of the DNA sequence of SEQ ID NO: 1 to 4. That is, the genotypes of the polymorphic regions of SEQ ID NOs: 1 to 4 are A / A (SEQ ID NO: 1), A / A (SEQ ID NO: 2), G / G (SEQ ID NO: 3), and A / A (SEQ ID NO: 4) If abnormal, the risk of premature menopause is high. The more the nucleic acid sequence having the risk genotype is detected in one sample, the higher the probability of belonging to the risk group can be determined.

The present invention also provides a nucleic acid sample from a sample, in order to provide information necessary for early menopause diagnosis; And (ii) analyzing haplotypes from the nucleic acid sample. Specifically, the haplotype analysis may be performed by analyzing the haplotype determined from the polymorphic site of the DNA sequence of SEQ ID NOs: 1 to 4 from the nucleic acid sample. The polymorphic sites of the DNA sequences of SEQ ID NOs: 1 to 4 refer to the 201st, 301th, 401th, and 301th bases, respectively.

That is, the haplotype analysis determined from the polymorphic site of the DNA sequence of SEQ ID NO: 1 to 4, the DNA sequence of SEQ ID NO: 1 to 4 located in the PCMT1 gene formed an associative non-equilibrium block, the DNA sequence of SEQ ID NO: 1 to 4 In the case of AAGA (ht1) haplotype, it can be determined that the risk of premature menopause is higher than that of those who do not, and in the case of GGAT (ht2), it can be determined to be resistant to premature menopause (Table 6, 1-2).

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

1. Selection of subjects

This study was conducted with 24 patients who were clinically diagnosed as early menopause from Korea Tea Hospital and their corresponding 24 normal subjects. In addition, experiments and analyzes were performed with consent from 84 patients and 170 normal subjects. They all have karyotypes of 46 and XX. All normal people were selected from people with children. The clinical criteria for early menopause were women under 40 years of age with FSH levels above 40 mIU / mL. The size of the clusters used is shown in Table 1 below.

group Normal Patient group total Stage 1 24 24 48 2 steps 194 108 302

2. Preparation of Genomic DNA

Blood was collected from premenopausal patients and 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 was 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) was 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 component 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 (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.

Fragmentation and Purification of Amplified Genomic DNA After the whole genome DNA amplification step, the cells were centrifuged at 50 g for 1 minute and transferred to three extra wells at 150 μL. 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.

Preparation and Sentrix ^R BeadChip (Sentrix ^R ) BeadChip was prepared while resuspending 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 transferred to the PB1 solution and shaken, and shaken at a time 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 (Hybridization Chamber), the genomic DNA was added, the BeadChip Chamber was put back into the hybridization chamber, and reacted by shaking at 48 ° C. at an appropriate speed for 18 hours.

After 18 hours of hybridization with Allele-Specific Primer Extension (ASPE) , the BeadChip chamber was removed and four 450 uL RA1 solutions were flowed. 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 the Illuminas 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 Illuminas 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

The Hardy-Weinberg equilibrium test was performed to determine the genetic distribution of the alleles that make up the monobasic polymorphisms. In this case, the criteria of HWE p value> 0.05 and Minor Allele Frequency (MAF)> 0.1, which are the criteria for polybasic polymorphism, were applied. Using the frequency of occurrence between the patient group and the normal group, the significance level of each monobasic polymorphism was set to <0.05 and analyzed using SAS software version 9.1.3. Correlation analysis was performed to compare the significant differences between the alleles and the frequency of occurrence in the patient and normal groups. The significance level was <0.05. Odds Ratio was calculated by logistic regression model and dominant, recessive and co-dominant models were applied. In analyzing the significance of each model, the majority allele is A1 and the minor allele is A2. The dominant model compares the sum of the frequency of A1A1 with the frequency of A1A2 and A2A2. The Recessive trait model compares the sum of A1A1 and A1A2 with the frequency of A2A2, while the Co-Dominant trait model compares the respective frequencies for A1A1, A1A2 and A2A2.

Associative non-equilibrium tests between monobasic polymorphs were performed. It used Haploview (ver. 4.1). Haplotypes were formed from associative non-equilibrium blocks formed in the analysis of the correlation between the single base polymorphisms in both the patient group and the normal group, and the patient-specific haplotypes were analyzed.

5. Results

(1) monobasic polymorphisms showing significance in the PCMT1 gene

Table 2 below shows the characteristics of the polymorphic sequences of SEQ ID NOs: 1 to 4 showing statistical significance as a result of analyzing the association between the normal group and the patient group and early menopause.

SEQ ID NO: Reference
SNP ID Allele location SNP Role Amino acid changes One rs4816 A> G 36508032 Missense Ile-> Val 2 rs2151913 A> G 33478979 Intron No change 3 rs7818 G> A 33498322 3'UTR No change 4 rs4552 A> T 33498322 3'UTR No change

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

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

Alleles represent major and minor alleles in each monobasic polymorph.

Position refers to the position of the standard base sequence on the chromosome (NCBI Genome build 36.3).

The SNP role indicates where the single base polymorphisms are located on the gene. 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 monobasic polymorphs are changing the amino acids constituting the protein when the gene is expressed to produce a protein.

(2) Genotype and Risk Allele Analysis

Tables 3a and 3b below show the allele frequencies of SEQ ID NOS: 1 to 4 and the frequencies of normal and patient groups for each genotype in the first and second analyzes. In addition, Tables 4a and 4b show the results of the Hardy-Weinberg Equilibrium Test and early menopause association analysis [i.e., Case-], which were divided first and second for genotype frequencies of Tables 3a and 3b, respectively. Case-control association study] As a result, it is possible to identify monobasic polymorphisms and their genotypes that may affect early menopause by the following association analysis.

In Table 3a and Table 3b, each column means as follows.

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

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

The allele frequency column indicates the two nucleotide bases that make up each monobasic polymorphism and indicates the frequency of each allele observed in the population. Here, an allele means a nucleotide sequence of nucleotides actually present at a single nucleotide polymorphism site, and a single nucleotide polymorphism may have two alleles due to its characteristics. According to the high and low frequency of these alleles, they are divided into large alleles and small alleles. In particular, the minor allele frequency (MAF) is used as a genetic reference for dividing a single nucleotide polymorphism and a point mutation based on 0.1. This criterion represents the stability of the polymorphism itself (that is, the site where a monobasic polymorphism occurs is constant), thus representing its potential use as a genetic marker.

Genotype consists of alleles that form each monobasic polymorphism, and shows what alleles are in the locus where the monobasic polymorphic site exists.

The genotype frequency column is made up of alleles that form a monobasic polymorphism and indicates what alleles are in the locus where the monobasic polymorphic site exists. Here, the genotype consisting of only large alleles is represented by the genotype consisting of the large major homotype, the large allele and the small allele is heterotyped, and the genotype consisting of only the small alleles is represented by the small minor homotype. It is called. The numbers represent the number of people of each genotype divided by normal and patient groups. This can be called an observation.

Tables 4a and 4b compare the frequency differences between the two alleles that form a single base polymorphism in the first and second phases of the study and the patient-normal population for the three types of genotypes that result. Analysis of which alleles or which genotypes are associated with the disease. This association analysis was carried out in four categories: the allele model, the co-dominant genotype, the dominant genotype, and the recessive model. The meaning of each column in Table 4a and Table 4b is as follows.

The sequence number is a number representing each monobasic polymorphism.

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

HWE represents the state of the Hardy-Weinberg Equilibrium. 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. This indicates whether the single marker polymorphisms observed in both premenopausal and normal groups in the first and second stages function as genetic markers and whether the distribution of genotypes is common.

The odds ratio (OR) represents the ratio of odds of risk alleles among normal groups to odds of risk alleles among patient groups. 95% CI represents the 95% confidence interval of the odds ratio, indicating (lower confidence interval, upper confidence interval). Confidence intervals with a value of 1 cannot be considered significant for association with disease. If the odds ratio is greater than 1 and does not include 1 in the 95% confidence interval, minor alleles have a higher frequency in the disease group. If the odds ratio is less than 1, the majority allele has a higher frequency in the disease group. Have

In the correlation analysis on the allele type, the Logit_p value represents the statistical significance for the odds ratio. This means the difference between the normal group and the patient group as much as the odds ratio. The criterion for determining that there is a significant difference is when the Logit_p value indicates a value of 0.05 or less. These values were obtained through logistic regression analysis.

ㆍ Exact p value in the correlation analysis of allele type also means the significance of the difference between the normal group and the patient group as the Logit_p value. This also recognizes a significant difference when the p value is less than 0.05. The Exact p value is obtained using a statistical method called Fisher's exact test, which was developed to perform a precise test when the expected frequency required for the comparison between the patient and the normal group is 5 or less. It is a statistical technique. Therefore, the data shown in Tables 4a and 4b are very suitable assays.

The risk allele was a small allele if the odds ratio was greater than 1 when the p-value was significant and a majority allele if the odds ratio was less than 1.

In analyzing the significance of the disease according to the four analysis models, if the majority allele is A1 and the minor allele is A2, the dominant model compares the sum of the frequency of A1A1 with the frequency of A1A2 and A2A2. The recessive trait model compares the sum of A1A1 and A1A2 with the frequency of A2A2, and the co-dominant trait model compares the respective frequencies for A1A1, A1A2 and A2A2.

Association analysis of alleles can determine how risk alleles are associated with disease through the proportion of frequencies in which alleles are present between patients and normal subjects. Since there is no significance when 1 is included in the 95% confidence interval, as can be seen from Tables 4A and 4B, the nucleotide sequences of the polymorphic regions of SEQ ID NOS: 1 to 4 are A, A, G, and A (risk), respectively. If one or more alleles are present, the risk of early menopause may be high.

In addition, SEQ ID Nos. 1 to 4 show significance in alleles, co-occurrence and dominant models, and SEQ ID Nos. 1 to 4 show minority alleles in persons having a genotype of Minor Allele with an odds ratio of <1. The risk of premature menopause was higher than those with major alleles, and those with two alleles of the genotype had a higher risk of premature menopause than those with at least one minor allele. appear.

From the above results, it can be determined that the polymorphic sites of SEQ ID NOs: 1 to 4 are at high risk of premature menopause if the genotype is one or more of A / A, A / A, G / G, and A / A. In addition, when one or more nucleotide sequences of the polymorphic regions of SEQ ID NOs: 1 to 4 are A, A, G, and A (risk alleles), respectively, it may be determined that there is a high possibility of early menopause. The more a nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to a risk group can be determined. Therefore, in the present invention, a single nucleotide polymorphism of SEQ ID NOs: 1 to 4 can be effectively used for diagnosis associated with early menopause. Specifically, it may be used as a target site for primers or probes for the diagnosis of early menopause.

(3) haploid analysis

Table 5 and FIG. 2 show the association between the polymorphic sites for SEQ ID Nos. 1 to 4, and FIG. 3 shows a haplotype formed from the Linkage Disequilibrium Block formed from FIG. (Haplotype). Table 6 shows the results of the association test with early menopause for haplotype of each block.

| D '| rs4816 rs2151913 rs7818 rs4552 r2 rs4816 - One One One rs2151913 0.942 - One One rs7818 0.944 0.889 - One rs4552 One 0.942 0.944 -

Table 5 shows the D 'values and the r ² value as shown by the dense quantify the association between the non-equilibrium block of each single nucleotide polymorphism shown in Fig. When the D 'value is more than 0.8, each of the single base polymorphisms is determined to be in an associative non-equilibrium relationship, and is shown in red as shown in FIG. These D 'values range from 0 to 1 and include calculations for the distance on genomic DNA between each monobasic polymorph. In addition, the r ² value represents the correlation between the single polymorphic polymorphisms. If the r ² value is 0.3 or more, it means that the single polymorphisms corresponding to the value are closely packed. This means that the aforementioned four monobasic polymorphisms are closely related to each other to form a genetic unit in FIG. 2 and Table 4 mentioned above. 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 an allele analysis of each monobasic polymorph, which is called a haplotype (FIG. 3).

FIG. 3 is a haplotype formed in comparison to genotyping results from an associative non equilibrium block formed from FIG. 2. Four haplotypes are shown from one of the associative non-equilibrium blocks shown in FIG. 2, and the haplotypes appearing specifically in the patient group and the haplotypes appearing in the normal group are observed in each block (Table 6). 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 allow us to predict not only the disease in the current generation, but also the transmission of the disease to the next generation. Table 6 is a table examining the significant difference in the frequency of the haplotypes of Figure 3 appear between the patient group and the normal group.

hts Haploid Total frequency Control POF Chi p-value Permutation
p-value ht1 AAGA 0.760 31 (64.6) 42 (87.5) 0.0085 0.0174 ht2 GGAT 0.219 15 (31.2) 6 (12.5) 0.0263 0.0298 ht3 AAAA 0.010 1 (2.1) 0 (0.0) 0.3148 One ht4 GAAT 0.010 1 (2.1) 0 (0.0) 0.3148 One

Two haplotypes were observed that showed significant differences between the patient and normal groups within one block formed. Haploid ht1 is shown as AAGA, which is high in the patient group. Haploid ht2, on the other hand, appears as GGAT, which is particularly high in the normal population. In conclusion, haplotype ht1 is a haplotype that is specific in the patient group. It can be predicted that those with haplotype have susceptibility to early menopause. In contrast, ht2 is a haplotype that is specific in the normal population, and it can be predicted that people with this haplotype are resistant to early menopause.

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. The single nucleotide polymorphism of the present invention appeared in a statistically significant level in early menopause patients, it can be seen that can be used for predictive diagnosis of early menopause from these monobasic polymorphism to the genetic unit.

Figure 1 shows the location of exons on the PCMT1 gene and the location of each monobasic polymorphism.

2 shows associative non-equilibrium blocks between monobasic polymorphs in the PCMT1 gene.

3 shows the constitution of haplotypes and respective monobasic polymorphs formed from associative non-equilibrium blocks.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Polynucleotides derived from PCMT1 gene comprising single          nucleotide polymorphism, microarrays and diagnostic kits          configuring the same, and analytic methods using the same <130> PN0242 <150> KR10-2008-0078292 <151> 2008-08-11 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Homo sapiens <400> 1 tgtttagcaa tctatagaag ccttaagcta tttttgtttt ctctttgtag aatatgactt 60 tcgataagga aaaacttcaa taatacttta gaaaaacaaa tttagcccaa tgagctactg 120 aattgttttc tcttttccag gttggatgta ctggaaaagt cataggaatt gatcacatta 180 aagagctagt agatgactca rtaaataatg tcaggaagga cgatccaaca cttctgtctt 240 cagggagagt acagcttgtt ggtaagtatc agaaaacttt aaaaatttct tcagaggatt 300 tttattttca gaagatccaa tgcaagctaa ataatactca gaggtaatta ttgtattttt 360 ttttacaatc acttttaagt actaaaaaat aaaaacgatt atttttgctc acctagtgtg 420 tattttgcag ccatctcttt gtgagtttaa ttttgtattt tcctattaac ctctgagatt 480 gaattttttg taatgtgtct gaccattagg attttgtctt ttgtgaagta cctgttcaga 540 tattggattg cttgtatttt tgacttacag ggcttctagc cctttgtcta tatatataaa 600 tacatttctt ctcccatctg aggtgtgcct tttcatctta ttggtaccct tttgataata 660 gaaattctta attttaatgt agtcagactt accaattttt t 701 <210> 2 <211> 601 <212> DNA <213> Homo sapiens <400> 2 atatgtatat atagaaagag gggaaagaaa tgtagaaaaa tgataataat tgatgactct 60 ggatgaagag atttaggagt tatttgtact atcctagcaa cttgttggca ataaaaatta 120 aagttaaaag ttttgaaaaa gttgttaaga taaatttgaa gggaagaaga aataaagagc 180 tcataatttc acaagtaagt gggggtaaat tactggacaa tttttttaaa ctaaattttg 240 taaattgtgc aagaaaatat ttttaaaatc atttctccaa taagctgcct ttatcctata 300 rtattggatt atctattaaa tgacaaatca taagtctgct tcctcttaat atattcttcc 360 aaataacaat gttataattc tcatagaaca gcttttactt aactcatgca ggcattaaaa 420 tatttggtca acaaagaatt atttgcaaag tgctctattc ataaagtaaa attcacttct 480 gctttttaga atatgaactg aagatattgt ttttcttgtt ttggcttttt tttttttttt 540 ttttttttga gacagagtct cgctctgtca cctaggctgg agtgcagtgg tgtgatcatg 600 g 601 <210> 3 <211> 801 <212> DNA <213> Homo sapiens <400> 3 aaagccaaat tgggtagtgt gtaactccta ctaatgtatt tctctctttt ttcctcacag 60 ggatgaattg taaaagcaac atcagcttga ccagtataaa attacagtgg attgctcatc 120 tcagtcctca aagctttttg aaaaccaaca ccatcacagc ttgttttgga ctttgttaca 180 ctgttatttt cagcatgaaa atgtgtgttt ttttagggtt tctgattctt caaagaggca 240 cagagccaaa ttggtagagg aaggatgcaa agtataaatt tgtgtaatat tactttaaca 300 tgcccatatt ttacttggaa atattaaaag aaagggttct gtaaaatgga aaacttagtt 360 tgtgaattga ttttgaggag tggtttttct tttcttggac rcttaattct gttctgatat 420 taatttatca gattgctttt gtgcattgga taacaccacc attcacaagt taagattctt 480 ggtatttgga tatctgttag atgctactaa gaaaatagag atgagctttc tttttaaagc 540 ttttgatgtg gtgtcataga atagcatgtt gtagatacaa tcagctgctt tgttacctta 600 aaactaggca tttgtaaata ttaaaccata agatggcagg tgatgtcctg taaacactca 660 gctgttcaga ttggacataa ctgacttagt tcttcccttc tctctctctc aaaattatag 720 gagacttgta gtttagtgtg gttttctgtt tctaatttgc tgctgataat gtatatgaac 780 ttaacatgct gtgtaagctg t 801 <210> 4 <211> 601 <212> DNA <213> Homo sapiens <400> 4 aagaaagggt tctgtaaaat ggaaaactta gtttgtgaat tgattttgag gagtggtttt 60 tcttttcttg gacacttaat tctgttctga tattaattta tcagattgct tttgtgcatt 120 ggataacacc accattcaca agttaagatt cttggtattt ggatatctgt tagatgctac 180 taagaaaata gagatgagct ttctttttaa agcttttgat gtggtgtcat agaatagcat 240 gttgtagata caatcagctg ctttgttacc ttaaaactag gcatttgtaa atattaaacc 300 wtaagatggc aggtgatgtc ctgtaaacac tcagctgttc agattggaca taactgactt 360 agttcttccc ttctctctct ctcaaaatta taggagactt gtagtttagt gtggttttct 420 gtttctaatt tgctgctgat aatgtatatg aacttaacat gctgtgtaag ctgtgtccta 480 gttcttgaac agtttatgca gtgctgcttt gccaaataaa gttaaaagta aaagaggcat 540 tggttgtttt tgaatataga aggaagaaca gcaagtccac cagataggga gtgtttacga 600 t 601  

Claims (11)

delete delete delete delete delete A polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, wherein the 201 base (polymorphic site) is A and is composed of 10 or more contiguous DNA sequences comprising the 201 base (a); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the 301th base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; And In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, a polynucleotide consisting of at least 10 consecutive DNA sequences comprising the 301th base (polymorphic site) A and comprising the 301th base; At least one selected from the group consisting of polynucleotides or complementary polynucleotides thereof, diagnostic kit for early menopause. In order to provide information necessary for early menopause diagnosis, (i) obtaining a nucleic acid sample from the sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from the polynucleotides of SEQ ID NOS: 1 to 4 or its complementary polynucleotides from the nucleic acid sample, provided that the polymorphic site is For the DNA sequence, the 201 base, the 301 base for the DNA sequence of SEQ ID NO: 2, the 401 base for the DNA sequence of SEQ ID NO: 3, and the 301 base for the DNA sequence of SEQ ID NO: 4). A method for detecting a monobasic polymorph in a sample. The method of claim 7, wherein the analyzing of the nucleotide sequence of the polymorphic site comprises a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NOs: 1 to 4 or a complementary polynucleotide sequence thereof comprising the polymorphic site; The polymorphic site is the 201 base for the DNA sequence of SEQ ID NO: 1, the 301 base for the DNA sequence of SEQ ID NO: 2, the 401 base for the DNA sequence of SEQ ID NO: 3, and the 301 base for the DNA sequence of SEQ ID NO: 4 A single base polymorphism in a sample, characterized in that carried out using a primer or probe with a template). The method of claim 7, wherein the analyzing the nucleotide sequence of the polymorphic site comprises at least 10 or more polynucleotides consisting of the DNA sequence of SEQ ID NO: 1, wherein the 201st base (polymorphic site) is A and comprises the 201st base. Polynucleotides (a) consisting of a contiguous DNA sequence; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the 301th base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, wherein the 301th base (polymorphic site) is A, and the polynucleotide (d) is composed of 10 or more consecutive DNA sequences comprising the 301th base. Hybridizing the nucleic acid sample to a microarray to which the polynucleotide or its complementary polynucleotide is immobilized; And analyzing the obtained hybridization result. 10. The method of claim 9, wherein the length of the DNA sequence of the polynucleotide immobilized on the microarray or its complementary polynucleotide is 10 to 100 nucleotides each. 8. The method of claim 7, wherein analyzing the nucleotide sequence of the polymorphic site is a polymorphic site of the DNA sequence of SEQ ID NO: 1, 2, 3, or 4, provided that the polymorphic site is the 201st for the DNA sequence of SEQ ID NO: Genotype or haplotype of base, 301 base for the DNA sequence of SEQ ID NO: 2, 401 base for the DNA sequence of SEQ ID NO: 3, and 301 base for the DNA sequence of SEQ ID NO: 4) A method for detecting a monobasic polymorph in a sample, characterized in that it comprises a (haplotype) analysis.

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