KR101196239B1 - Diagnostic kit for premature ovarian failure comprising polynucleotides having single nucleotide polymorphism derived from TG gene and analytic method using the same - Google Patents

Abstract

The present invention provides a kit for diagnosing early menopause in Koreans comprising a polynucleotide comprising a mononucleotide polymorphism derived from the TG gene or a complementary polynucleotide thereof; And an analysis method for providing information necessary for early menopause diagnosis in Korean using the polynucleotide or its complementary polynucleotide. Such polynucleotides or complementary polynucleotides thereof may be used as markers for the clinical diagnosis of premature menopause.

Description

Diagnostic kit for premature ovarian failure comprising polynucleotides having single nucleotide polymorphism derived from TG gene and analytic method using the same}

The present invention includes a polynucleotide comprising a single-nucleotide polymorphism (SNP) derived from a TG (Thyroglobulin) gene or a kit for diagnosing early menopause in a Korean including a complementary polynucleotide thereof; And an analysis method for providing information necessary for diagnosing Premature Ovarian Failure (POF) in Korean 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 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 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 the single base polymorphisms selected through linkage equilibrium analysis are at linkage equilibrium, the alleles of the single base polymorphism are removed because the genetic changes in the single base polymorphisms are at the same linkage equilibrium. Analysis reveals the presence of mutations in 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, and found a monobasic polymorphism site specific to premenopausal patients from the TG (Thyroglobulin) gene. The present invention has been completed.

Accordingly, an object of the present invention is to provide a kit for diagnosing early menopause in a Korean including a single base polymorphism derived from the TG gene as a single base polymorphism associated with early menopause.

It is also an object of the present invention to provide a method for early menopause diagnosis in Korean 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 polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 201 base (polymorphic site) is G, and comprising said 201 base ( a); A polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, wherein the 201 base (polymorphic site) is G and the polynucleotide (b) is composed of 10 or more consecutive DNA sequences comprising the 201 base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 10, wherein the 344th base (polymorphic site) is C and is one from the group consisting of polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 344th base Provided is a kit for diagnosing early menopause in Korean, comprising the polynucleotide selected above or its complementary polynucleotide.

In addition, according to another aspect of the present invention, to provide information necessary for the diagnosis of early menopause in Korean, (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, 2, and 10 or complementary polynucleotides thereof from the nucleic acid sample, wherein the polymorphic site is SEQ ID NO: Single base polymorphism in the sample, including 201 base for DNA sequence 1, 201 base for DNA sequence 2, and 344 base for DNA sequence 10; A method is provided.

It has been found by the present invention that at least one polynucleotide selected from the group consisting of polynucleotides (a) to (c) or complementary polynucleotides thereof can be usefully used for the diagnosis of early menopause in Koreans. Therefore, the kit comprising the polynucleotide or its complementary polynucleotide can be usefully used for predictive diagnosis of early menopause in Koreans; The analysis method of the present invention using the polynucleotide or its complementary polynucleotide can be usefully used to provide information necessary for early menopause diagnosis in Koreans.

1 shows the associative unbalance block of the TG gene.
2 shows haplotypes formed from associative unbalance blocks of the TG gene.

The present invention relates to a polynucleotide or complementary polynucleotide selected from the group consisting of polynucleotides (a) to (c) derived from the sequences of SEQ ID NOs: 1, 2 and 10 newly found to be associated with premature menopause in Koreans. It provides a kit for diagnosing early menopause in Korean. 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 G, and the polynucleotide is composed of 10 or more consecutive DNA sequences including the 201 base; A polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, wherein the 201 base (polymorphic site) is G and the polynucleotide (b) is composed of 10 or more consecutive DNA sequences comprising the 201 base; In the polynucleotide consisting of the DNA sequence of SEQ ID NO: 10, At least one from the group consisting of polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 344th base (C polymorphic site) is C, the 344th base Provided is a kit for diagnosing early menopause in a Korean including a selected polynucleotide or a complementary polynucleotide thereof.

The DNA sequences of SEQ ID NOs: 1, 2, and 10 are single nucleotide polymorphisms showing statistically significant difference (significance level p value <0.05) in the premenopausal patient group and the control group. exist.

The present inventors conducted various studies to select TG as a candidate gene associated with early menopause and to develop diagnostic indicators at the molecular level in early menopause diagnosis. We performed genotyping and statistical analysis on 98 Korean controls and 218 premenopausal patients. Only a few of the single nucleotide polymorphisms showed statistically significant differences between the control and patient groups. I found that. In particular, the single nucleotide polymorphisms of SEQ ID NOs: 1, 2, and 10 confirmed the presence of a risk allele at each polymorphic site as a result of Fisher's exact assay and permutation assay for correction of multiple comparisons.

The diagnostic kit according to the present invention may include a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (c) or a complementary polynucleotide thereof; The length of the polynucleotide or its complementary polynucleotide included in the diagnostic kit according to the present invention may be 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 (c) or complementary polynucleotides thereof 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 is a site in a polymorphic sequence where a single base polymorphism occurs. The polynucleotide can be DNA or RNA.

The present invention also includes an allele-specific primer for amplification for early menopause diagnosis, comprising at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (c) 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 single nucleotide polymorphism of SEQ ID NO: 1, 2 and 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 can be used by other methods as well as GoldenGate Assay.

The diagnostic kit according to the present invention comprises a microarray form comprising the polynucleotide or its complementary nucleotides as a probe, that is, at least one polynucleotide selected from the group consisting of polynucleotides (a) to (c) or a complementary polynucleotide thereof. It may be in the form of a microarray including as a probe.

The present invention to provide information necessary for the early diagnosis of menopause in Korean, (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, 2, and 10 or complementary polynucleotides thereof from the nucleic acid sample, wherein the polymorphic site is SEQ ID NO: Single base polymorphism in the sample, including 201 base for DNA sequence 1, 201 base for DNA sequence 2, and 344 base for DNA sequence 10; It includes how to. Preferably, step (ii) may be performed by analyzing the nucleotide sequence of the polymorphic site of the polynucleotide of SEQ ID NO: 2 or its complementary nucleotide from the nucleic acid 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 salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA) and Nonidet P-40. After the addition of the solution, the resulting solution was mixed well and centrifuged at about 2,200 rpm for 10 minutes at room temperature, and the resulting mass was concentrated in a high-salt buffer solution (10 mM Tris-HCl [pH 7.6]). , 10 mM KCl, 10 mM MgCl ₂ , 0.4 M NaCl, and 2 mM EDTA) Genomic DNA is mixed with 50ul of 10% SDS and reacted overnight at about 55 ° C. After the reaction, each genomic DNA is 1.5 ml Transfer to a volume centrifuge test tube, add 0.3 ml of 6 M NaCl, mix genomic DNA well, and centrifuge for 10 minutes at approximately 12,000 rpm After centrifugation, collect the upper layer containing genomic DNA and transfer to a new test tube. Equivalent amount of 100% ethanol is added at room temperature. Mix well until washed, and wash genomic DNA with 70% ethanol when settled, open the test tube lid, allow the solution to dry, and add TE buffer (pH 8.0) to reproduce the genomic DNA. By turbidity, a nucleic acid sample can be obtained.

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, 2 and 10 (except the polymorphism) The site means a primer or probe having a template of 201 base for the DNA sequence of SEQ ID NO: 1, 201 base for the DNA sequence of SEQ ID NO: 2, and 344 base for the DNA sequence of SEQ ID NO: 10). It can be performed using. 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 to which a polynucleotide selected from the group consisting of polynucleotides (a) to (c) or a complementary polynucleotide thereof is immobilized. Wherein the polynucleotides (a) to (c) are as defined above); And analyzing the obtained hybridization results. Preferably, the step of analyzing the nucleotide sequence of the polymorphic site, in the polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, the 20th base (polymorphic site) is G, 10 or more consecutive DNA sequences comprising the 201st base Hybridizing the nucleic acid sample to a microarray to which a polynucleotide consisting of: or a complementary nucleotide thereof is immobilized; And analyzing the obtained hybridization results. The DNA sequence of the polynucleotide or its complementary nucleotides immobilized on the microarray may be 10 to 100 nucleotides each.

The results obtained according to the method for detecting a monobasic polymorphism in a sample of the present invention may provide information necessary for early menopausal diagnosis in Korean, for example, the bases of the polymorphic sites of If there is more than one G, C (risk allele), it can be determined that they belong to a risk group with a 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.

In addition, analyzing the base sequence of the polymorphic site may include genotype analysis of the polymorphic site of the DNA sequence selected from the group consisting of DNA sequences of SEQ ID NOs: 1, 2 and 10. That is, the genotypes of the polymorphic regions of SEQ ID NOs: 1, 2, and 10 are G / G (SEQ ID NO: 1), G / G and A / G (SEQ ID NO: 2), and C / C and A / C (SEQ ID NO: 10) If more than one is present, the risk of early menopause may be 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, in order to provide information necessary for early menopause diagnosis in Korean, (i) obtaining a nucleic acid sample from the sample; 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: 2-9 from the nucleic acid sample. The polymorphic sites of the DNA sequence of SEQ ID NO: 2 to 9 are each of the 201 base of SEQ ID NO: 2, the 201 base of SEQ ID NO: 3, the 301 base of SEQ ID NO: 4, the 444 base of SEQ ID NO: 5, and the sequence of 6 The 301th base, the 201st base of SEQ ID NO: 7, the 301th base of SEQ ID NO: 8, and the 301th base of SEQ ID NO.

That is, the haplotype analysis determined from the polymorphic site of the DNA sequence of SEQ ID NOs: 2 to 9 indicates that, if the DNA sequence of SEQ ID NOs: 2 to 9 has the GGCCCTAT haplotype, the risk of premature menopause is higher than that of the other. Can be. (See Table 7, FIGS. 1 and 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 performed with the consent of 98 patients who were clinically diagnosed as early menopause from Korea Tea Hospital and the corresponding 218 controls. The control group had a normal menstrual cycle before menopause and was selected from women who experienced natural pregnancy. The clinical diagnostic criteria for early menopause was women under 40 years of age with FFS levels above 40 IU / L. The size of the clusters used is shown in Table 1 below.

group Control group Patient group total Survey 218 98 316

2. Preparation of Genomic DNA

Blood was collected from premenopausal patients and control 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 5 ml of low concentration salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA). Nonidet P-40 was added, and the resulting solution was mixed well, and then centrifuged at 2,200 rpm for 10 minutes at room temperature The resulting mass was a high concentration salt 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 GoldenGate Assay was adjusted to a concentration of 250ng / 5uL.

3. Genotyping of the whole genome-GoldenGate assay

According to the GoldenGate assay method, genotyping was performed using a Sentrix array matrix chip. All reagents and Sentrix array matrix chips for GoldenGate assay were purchased from Illumina Inc. All procedures were performed according to Illumina's GoldenGate assay method.

Genomic DNA samples (250 ng / 5 μl) were mixed with 5 ul of GS # -MS1 reagent in 96 well plates. The plate was sealed and centrifuged for 1 minute at 250 × g, and reacted for 30 minutes in a 95 ° C. heat block. 5 ul GS # -SUD was added thereto and mixed, 5 ul 2-propanol was added thereto, and mixed again, followed by centrifugation at 3,000 × g for 20 minutes, and then the supernatant was removed and the pellet was dried. The dried DNA was added well with 10 ul of GS # -RS1 reagent. 10 ul of GS # -OPA and 30 ul of GS # -OB1 were added thereto, mixed well, and placed in a 70 ° C. heat block and slowly cooled down to 30 ° C. The GS # -ASE plate was placed on the magnetic plate and left for 2 minutes. The supernatant was removed and 50 ul GS # -AM1 was added and mixed well. After placing on a magnetic plate and left for 2 minutes, the supernatant was removed, 50 ul GS # -AM1 was added and mixed well. After washing with 50 ul GS # -UB1 in the same manner as above, the supernatant was removed, 37 ul GS # -MEL was added and well mixed and incubated for 15 minutes at 45 ℃. The upper plate was placed again on the magnetic plate, left for 2 minutes, the supernatant was removed, and 50 ul GS # -UB1 was added thereto. Repeat the above process again, put 35 ul GS # -IP1 and mix well and incubated for 1 minute in a 95 ℃ heat block. The plate was placed on a magnetic plate and left for 2 minutes. 30 ul of the supernatant was transferred to the GS # -PCR plate, 64 ul of illumina-recommended DNA polymerase and 100 ul UDG were added to the GS # -MMP tube, mixed well, and amplified under the conditions shown in Table 2.

Temperature Time at each temperature 37 ℃ 10 min 95 ℃ 3 min 34 cycles 95 ℃ 35 sec 56 ℃ 35 sec 72 ℃ 2 min 72 ℃ 10 min 4 ℃ 5 min

After the reaction, 20 ul of GS # MPB was added and mixed well, and then transferred to a 96 well filter plate, and left in the dark for 60 minutes. The filter plate adapter was placed in a 96 well V-bottom and centrifuged to remove the supernatant. 50 ul GS # -UB2 was put back into the filter plate and centrifuged at 1000 x g for 5 minutes. A GS # -INT plate containing 30 ul of GS # _MH1 was placed on the filter plate and 30 ul of 0.1N NaOH was placed in the filter plate. PCR products were harvested by centrifugation at 1000 xg for 5 minutes.

The chips were pretreated in GS # -UB2 and NaOH, and the collected PCR products were transferred to 384 wells and the pretreated chips were loaded thereon. Hybridization was performed at 60 ° C. for 30 minutes and again the temperature was changed to 45 ° C. and hybridized for at least 14 hours. After washing in GS # UB2, GS # IS1, and dried at room temperature, image scanning was performed for analysis.

Genotyping Analysis of genotypes from image files confirmed the genotype for each polymorphism using Illumina's Beadstudio. Beadstudio exhibits a typical genotype pattern with a ratio of the intensity of color development between probes recognizing two alleles.

4. Statistical Analysis

The Hardy-Weinberg equilibrium test was performed to determine the genetic distribution of the alleles that make up the monobasic polymorphisms. Here, the criteria of HWE p value> 0.05 and Minor Allele Frequency (MAF)> 0.05, which are the criteria for polybasic polymorphism, were applied. Using the frequency of occurrence between the patient and control group, the significance level of each polybasic polymorphism was set at <0.05 and analyzed using SAS software version 9.1.3 and PLINK program 1.07. Correlation analysis showed significant differences between alleles in the patient and control 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 the disease according to each model, the allele having a higher frequency in the disease group than the control group was A1, and the allele having a higher frequency in the control group than the disease group was A2. The dominant model compares the sum of the frequencies of A1A1 and A1A2 with the frequency of A2A2, and the recessive trait model compares the sum of the frequencies of A1A1 with the frequency of A1A2 and A2A2. Co-Dominant trait model is to compare the frequency of each for A1A1, A1A2 and A2A2.

5. Results

(1) monobasic polymorphisms showing significance in the TG gene

Table 3 below shows the characteristics of the polymorphic sequences of SEQ ID NOs: 1 to 10 showing statistical significance as a result of analyzing the association between the control group and the patient group and early menopause.

SEQ ID NO: Reference
SNP ID Allele location SNP Role amino acid
change One rs16904774 G> A 133900495 missense G → R 2 rs2069556 A> G 133920518 missense D → G 3 rs2687810 A> G 133935292 intron No change 4 rs1474539 T> C 133946089 intron No change 5 rs1879991 T> C 133958037 intron No change 6 rs2246625 T> C 133960936 intron No change 7 rs2246608 C> T 133961279 intron No change 8 rs2069561 G> A 133975283 missense D → N 9 rs2687814 C> T 133975406 intron No change 10 rs3758113 A> C 134034613 intron 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 Alleles 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

Table 4 below shows the allele frequencies of the DNA sequences of SEQ ID NOs: 1 to 10 and the frequencies of the control and patient groups for each genotype. Table 5 also shows the results of the Hardy-Weinberg Equilibrium Test and early menopause association analysis (ie, Case-control association study) for genotype frequencies. It is possible to identify single nucleotide polymorphisms (ie, single nucleotide polymorphisms of SEQ ID NOs: 1, 2, and 10) and genotypes thereof that may affect early menopause.

In Table 4, the meaning of each column is 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 major alleles and minor 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 0.01. 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 and genotype frequency% column is made up of alleles that form a monobasic polymorphism, and shows what alleles are in the locus where the monobasic polymorphic site exists. Here, the genotype consisting of a large number of alleles is represented by a majority homotype, a genotype consisting of a large number of alleles and a minor allele is a heterotype, and a genotype consisting of a minor allele is a minor homotype. It is called. The numbers represent the number of people of each genotype divided by the control and patient groups. This can be called an observation.

Table 5 compares the frequency differences between the patient-control groups for the three types of genotypes formed for one single polymorphism and analyzes 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. Meaning of each column in Table 5 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 that the function as a genetic marker of each monobasic polymorphism observed in both premenopausal patients and control groups, and whether the distribution of genotypes consisting of them is common.

Odds ratio (OR) represents the ratio of odds of risk alleles in the control group to odds of risk alleles in the patient group. 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.

Permutation p value in the correlation analysis for each analysis model means the significance of the difference between the control group and the patient group. The criterion to judge that there is a significant difference is when the permutation p value indicates a value of 0.05 or less. These values were obtained through a permutation test analysis. In Fisher's exact test results, the correction of the multiple comparisons is the Permutation p value.

The risk allele was a case where the odds ratio was greater than 1 when any of the four analytical models had a significant permutation p value.

ㆍ In analyzing the significance of disease according to each of the three analytical models, the allele having a higher frequency in the disease group than the control group is A1 and the allele having a higher frequency in the control group than the disease group is A2. Is a comparison of the frequency of A1 and A2 in the disease group and the control group, the dominant model compares the sum of the frequencies of A1A1 and A1A2 with the frequency of A2A2, and the recessive trait model is the frequency of A1A1. The sum of the frequencies of A1A2 and A2A2 is compared, and the Co-Dominant trait model compares the respective frequencies for A1A1, A1A2 and A2A2.

The allele can determine how risk alleles are associated with disease through the ratio of the frequency at which alleles appear between patient and control groups. Since there is no significance when 1 is included in the 95% confidence interval, as shown in Table 5, the nucleotide sequences of the polymorphic sites of SEQ ID NOs: 1, 2, and 10 are G, G, and C (risk alleles), respectively. If more than one is present, the risk of early menopause is high.

In addition, SEQ ID NO: 1 shows significance in allele and recessive models, and the risk of premature menopause in people with genotypes of major allele is greater than those with minor alleles. SEQ ID NO: 2 is significant in the dominant model, and those with genotype Minor Allele have early menopause than those with Major Allele. The risk of catching was higher, and those who had at least one minor allele had a higher risk of premature menopause, and SEQ ID NO: 10 was significant in allelic and dominant models and was genotype. People who have this minor allele are at risk for early menopause than those who have major allele More was found to be high, it showed that people who have any of the minority allele higher risk of early menopause.

From the above results, the genotypes of the polymorphic regions of SEQ ID NOs: 1, 2 and 10 were G / G (SEQ ID NO: 1), G / G and A / G (SEQ ID NO: 2), C / C and A / C (SEQ ID NO: 10 ) May be considered to be at high risk of premature menopause, and at least one nucleotide sequence of the polymorphic regions of SEQ ID NOs: 1, 2, and 10 is G, G, and C (risk allele), respectively. If present, the risk of premature menopause is high. 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, the single nucleotide polymorphisms of SEQ ID NOs: 1, 2, and 10 can be effectively used for diagnosis related to 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 6 and FIG. 1 show the association between the polymorphic sites for SEQ ID NOs 2-9, and FIG. 2 shows the haplotype formed from the Linkage Disequilibrium Block formed from FIG. 1 based on this. Table 7 shows the results of the association test with early menopause for haplotypes in the block.

Table 6 quantifies the compactness of the associative non-equilibrium blocks between the monobasic polymorphs shown in FIG. 1, and shows the values of D ′ and r ² . When the D 'value is more than 0.8, each of the monobasic 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 of 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 in the aforementioned FIG. 1 and Table 6, the eight 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 an allele analysis of each monobasic polymorph, which is called a haplotype (FIG. 2).

FIG. 2 is a haploid form formed from an associative unbalanced block formed from FIG. Three haplotypes are shown from one of the associative non-equilibrium blocks shown in FIG. 2. Among them, except for the haplotypes of which frequency is less than 0.1, two haplotypes are analyzed. It was observed to appear (Table 7). In addition, SEQ ID NOs: 2 and 5 were identified as tag SNPs to represent other SNPs in the block. 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 7 examines whether one haplotype shows a significant difference in the frequency of appearance between the patient group and the normal group. One haplotype was observed that showed a significant difference between the patient group and the normal group within one block formed. The haplotype is G-G-C-C-C-T-A-T, which is significant in the dominant model and has a particularly high frequency in the patient group. In conclusion, the haplotypes that are specific to the haploid G-G-C-C-C-T-A-T patient group can be expected to have susceptibility to early menopause.

Attach an electronic file to a sequence list

Claims (8)

In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, the 201 base (polymorphic site) is G, and comprises a polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 201 base or a complementary nucleotide thereof, Kit for Diagnosing Early Menopause in Koreans. The kit for diagnosing early menopause in Korean according to claim 1, wherein the polynucleotide or its complementary nucleotide is 10 to 100 nucleotides in length. The kit for diagnosing early menopause in Korean according to claim 1, wherein the kit has a microarray form comprising the polynucleotide or its complementary nucleotide as a probe. In order to provide information necessary for early menopause diagnosis in Korean, (i) obtaining a nucleic acid sample from the sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of the polynucleotide of SEQ ID NO: 2 or its complementary nucleotide from the nucleic acid sample, wherein the polymorphic site refers to the 201 base of the DNA sequence of SEQ ID NO: 2). A method for detecting a monobasic polymorph in a sample comprising a. The sequence of claim 4, wherein analyzing the nucleotide sequence of the polymorphic site comprises a polynucleotide of SEQ ID NO: 2 or a complementary polynucleotide sequence thereof comprising the polymorphic site, wherein the polymorphic site is a DNA sequence of SEQ ID NO: 2. Method of detecting a single nucleotide polymorphism in a specimen, characterized in that performed using a primer or a probe with a template). The method of claim 4, wherein the analyzing the base sequence of the polymorphic site comprises: 10 polynucleotides comprising the DNA sequence of SEQ ID NO: 2, wherein the 201th base (polymorphic site) is G and the 201th base comprises the 201 base. Hybridizing the nucleic acid sample to a microarray having a polynucleotide consisting of the above contiguous DNA sequences or a complementary nucleotide thereof fixed thereto; And analyzing the obtained hybridization result. The method of claim 6, wherein the DNA sequence of the polynucleotide or the complementary nucleotide thereof fixed to the microarray is 10 to 100 nucleotides in length. The method of claim 4, wherein analyzing the nucleotide sequence of the polymorphic site comprises genotype analysis of the polymorphic site of the DNA sequence of SEQ ID NO: 2. 6.

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