KR101196241B1 - Analytic method for diagnosing premature ovarian failure using polynucleotides comprising single nucleotide polymorphism derived from HSD17B4 gene and analytical kit therefor - Google Patents

Abstract

The present invention provides information necessary for early menopause diagnosis in Korean comprising analyzing a polynucleotide comprising a monobasic polymorphism derived from the HSD17B4 gene or a haplotype determined from its complementary polynucleotide. Analytical method; And it provides an analysis kit for this.

Description

Analytical method for diagnosing premature ovarian failure using polynucleotides comprising single nucleotide polymorphism derived from HSD17B4 gene and analytical kit therefor }

The present invention provides a method for analyzing a haplotype determined from a polynucleotide comprising a single-nucleotide polymorphism (SNP) derived from a HSD17B4 (hydroxysteroid (17-beta) dehydrogenase 4) gene or a complementary polynucleotide thereof. Analytical methods to provide information necessary for the diagnosis of Premature Ovarian Failure (POF) in Koreans; And it relates to an analysis kit for this.

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 single nucleotide polymorphism specific to early menopause in women, especially Korean women, and showed that the HSD17B4 (hydroxysteroid (17-beta) dehydrogenase 4) gene is specific to early menopause patients. The monobasic polymorphic site was searched. As a result, alleles and genotypes of all monobasic polymorphisms tested showed no statistical significance between premenopausal patients and normal subjects, but surprisingly the haplotype determined by the combination of specific monobasic polymorphisms was found between premenopausal patients and normal subjects. It was found to indicate statistical significance.

Accordingly, the present invention provides an analysis for providing information necessary for early menopause diagnosis in Korean, comprising analyzing a polynucleotide comprising a monobasic polymorphism derived from the HSD17B4 gene or a haplotype determined from its complementary polynucleotide. It is an object to provide a method.

In addition, an object of the present invention is to provide an analysis kit usefully used in the above analysis method.

According to one aspect of the invention, in order to provide information necessary for early menopause diagnosis in Korean, (i) obtaining a nucleic acid sample from a sample; And (ii) analyzing the haplotype determined from the polymorphic site of the DNA sequence of SEQ ID NOS: 1-5 from the nucleic acid sample, provided that the polymorphic sites of the DNA sequence of SEQ ID NOS: 1-5 are Single nucleotide polymorphism in a sample, comprising a 251 base, 201 base of SEQ ID NO: 2, 1117 base of SEQ ID NO: 3, 301 base of SEQ ID NO: 4, and 501 base of SEQ ID NO: 5) A method is provided.

Furthermore, according to another aspect of the present invention, in the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the polynucleotide consisting of ten or more consecutive DNA sequences comprising the 251 st base (polymorphic site) is A and comprising the 251 st base. Nucleotide (a) or a complementary polynucleotide thereof; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) or a complementary polynucleotide consisting of 10 or more consecutive DNA sequences including the 201 base (G) and G comprising the 201 base ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 1117 th base (polymorphic site) is T, and the complementary polynucleotide thereof ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the polynucleotide (d) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the complementary polynucleotide thereof ; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, wherein the 501th base (polymorphic site) is C, the polynucleotide (e) consisting of 10 or more consecutive DNA sequences comprising said 501th base, or a complementary poly There is provided a haplotype analysis kit comprising nucleotides, which is determined from the polymorphic site of the DNA sequence of SEQ ID NOs. 1 to 5 for early menopause diagnosis in Koreans.

The DNA sequences of SEQ ID NOs: 1 to 5 derived from the HSD17B4 gene form one block to form a haplotype, and the haplotype shows a statistically significant difference between premenopausal patients and normal people in Korea. Revealed by the invention. Therefore, the haplotype analysis of the DNA sequences of SEQ ID NOs: 1 to 5 can be usefully used to provide information necessary for early menopause diagnosis in Koreans; An analytical kit comprising the polynucleotide or its complementary polynucleotide can be usefully used in the assay method.

1 shows an associative unbalance block of the HSD17B4 gene.
2 shows haplotypes formed from associative unbalance blocks of the HSD17B4 gene.

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 haplotype determined from the polymorphic site of the DNA sequence of SEQ ID NOS: 1-5 from the nucleic acid sample, provided that the polymorphic sites of the DNA sequence of SEQ ID NOS: 1-5 are Single nucleotide polymorphism in a sample, comprising a 251 base, 201 base of SEQ ID NO: 2, 1117 base of SEQ ID NO: 3, 301 base of SEQ ID NO: 4, and 501 base of SEQ ID NO: 5) Provide a way to.

The DNA sequences of SEQ ID NOs: 1 to 5 did not show statistically significant differences in alleles and genotypes in the premenopausal patients and the control group, but the haplotypes formed from them showed a single base polymorphism showing a statistically significant difference. As such, they are present in the HSD17B4 gene located on chromosome 5.

The present inventors have selected HSD17B4 as a candidate gene associated with early menopause, and conducted various studies to develop diagnostic indicators at the molecular level in early menopause diagnosis. We performed genotyping and statistical analysis on 98 Korean controls and 214 premenopausal patients. Only a few polybasic polymorphic polymorphisms showed statistically significant differences between the control and patient groups. I found that. In particular, the haplotype consisting of SEQ ID NOs: 1 to 5 was found to show statistically significant differences between the control group and the patient group as a result of Fisher's exact test and correction of the multiple comparisons.

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.

Analyzing the haplotype can be performed using a primer or probe whose template is a polynucleotide of SEQ ID NOs: 1 to 5 or a complementary polynucleotide sequence thereof. That is, the step of analyzing the haplotype is a sequence comprising a polymorphic site as a polynucleotide of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 251 base of the DNA sequence of SEQ ID NO: ; A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 2 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 201 base of the DNA sequence of SEQ ID NO: 2; A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 1117th base of the DNA sequence of SEQ ID NO: 3; A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 4 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 301th base of the DNA sequence of SEQ ID NO: 4; And primers or probes each having a polynucleotide of SEQ ID NO: 5 or a sequence comprising a polymorphic site as a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 501th base of the DNA sequence of SEQ ID NO: 5 This can be done through sequencing of each polymorphic site used. The primer or probe may be prepared from the sequence in a conventional manner through conventional methods.

In addition, the step of analyzing the haplotype may hybridize the nucleic acid sample to a microarray having a polynucleotide derived from a DNA sequence of SEQ ID NOs: 1 to 5 or a complementary polynucleotide thereof, and to analyze the hybridization result obtained therefrom. It can be performed including a step. That is, the step of analyzing the haplotype is a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the 251 st base (polymorphic site) is A, the poly consisting of 10 or more consecutive DNA sequences comprising the 251 st base Nucleotide (a) or a complementary polynucleotide thereof; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) or a complementary polynucleotide consisting of 10 or more consecutive DNA sequences including the 201 base (G) and G comprising the 201 base ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 1117 th base (polymorphic site) is T, and the complementary polynucleotide thereof ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the polynucleotide (d) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the complementary polynucleotide thereof ; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, wherein the 501th base (polymorphic site) is C, the polynucleotide (e) consisting of 10 or more consecutive DNA sequences comprising said 501th base, or a complementary poly Hybridizing the nucleic acid sample to a nucleotide-fixed microarray; And analyzing the obtained hybridization results to determine the base sequence of the polymorphic site. The length of the DNA sequence of the polynucleotide or its complementary nucleotide fixed to the microarray may be 10 to 100 nucleotides, preferably 20 to 60 nucleotides, more preferably 40 to 60 nucleotides, respectively.

Results obtained according to the method for detecting a single nucleotide polymorphism in a sample of the present invention can provide information necessary for early menopausal diagnosis in Korean, and the haplotype analysis result determined from the polymorphic sites of SEQ ID NOs: 1 to 5, SEQ ID NO: 1 When the DNA sequence of 5 to 5 has an AGTAC haplotype, it may be determined that the risk of premature menopause is low since it is resistant to early menopause (see Table 7, FIGS. 1-2).

The present invention also provides a haplotype analysis kit useful for the above assay method. That is, the present invention is a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, polynucleotide (a) consisting of ten or more consecutive DNA sequences comprising the 251 st base (polymorphic site) is A, the 251 st base or Its complementary polynucleotides; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) or a complementary polynucleotide consisting of 10 or more consecutive DNA sequences including the 201 base (G) and G comprising the 201 base ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 1117 th base (polymorphic site) is T, and the complementary polynucleotide thereof ; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the polynucleotide (d) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the complementary polynucleotide thereof ; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, wherein the 501th base (polymorphic site) is C, the polynucleotide (e) consisting of 10 or more consecutive DNA sequences comprising said 501th base, or a complementary poly Provided is a haplotype analysis kit including nucleotides, which is determined from a polymorphic site of the DNA sequence of SEQ ID NOs. 1 to 5 for early menopause diagnosis in Koreans.

The assay kit according to the present invention may comprise a polynucleotide hybridizing with each of the polynucleotides of the polynucleotides (a) to (e) or each of its complementary polynucleotides. In addition, the polynucleotide (a) or a complementary nucleotide thereof; Polynucleotide (b) or a complementary nucleotide thereof; Polynucleotide (c) or a complementary nucleotide thereof; Polynucleotide (d) or a complementary nucleotide thereof; And the length of the polynucleotide (e) or its complementary nucleotides may be 10 to 100 nucleotides, preferably 20 to 60 nucleotides, more preferably 40 to 60 nucleotides.

The assay kit according to the present invention comprises a microarray form comprising the polynucleotide or its complementary nucleotides as a probe, that is, the polynucleotide (a) or its complementary nucleotides; Polynucleotide (b) or a complementary nucleotide thereof; Polynucleotide (c) or a complementary nucleotide thereof; Polynucleotide (d) or a complementary nucleotide thereof; And a microarray form comprising a polynucleotide (e) or a complementary nucleotide thereof as a probe, respectively.

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 98 patients who were clinically diagnosed as early menopause from Korea Tea Hospital and their corresponding 214 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 214 98 312

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. In this case, the criteria of HWE p value> 0.05 and Minor Allele Frequency (MAF)> 0.05, which are the criteria for polymorphism, were used. The correlation between the haplotypes for the disease was set at <0.05, using the frequency of occurrence between the patient and the control group, and analyzed using SAS software version 9.1.3 and PLINK program version 1.07. Correlation analysis showed significant differences between the haplotype patient group and the control group. The significance level was <0.05. Odds Ratio was calculated through the logistic regression model, dominant (Dominant, + / + plus +/- vs.-/-), recessive (+ / + vs. +/- plus-/-) and Co-Dominant (+ / + vs. +/- vs.-/-) model was applied.

5. Results

(1) monobasic polymorphisms showing significance in HSD17B4 gene

Table 3 shows the results of analyzing the association between the control group and the patient group, and showed no statistical significance in the allele and the genetic analysis, but showed the statistical significance in the haplotype analysis. Characterization of polymorphic sequences is shown.

SEQ ID NO: Reference
SNP ID Allele location SNP Role amino acid
change One rs3756512 A> C 118812383 intron No change 2 rs3797360 G> A 118825343 intron No change 3 rs32665 C> T 118847140 intron No change 4 rs3797368 A> G 118859958 intron No change 5 rs1490756 C> T 118874287 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 frequency of minor alleles of the DNA sequences of SEQ ID NOs: 1-5, the results of the Hardy-Weinberg Equilibrium Test and the frequency of the control and patient groups for each genotype.

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 Minor allele frequency column indicates the frequency of the less frequent allele of the two nucleotide bases constituting each monobasic polymorph. The frequency of minor alleles is used as a genetic criterion for dividing single base polymorphisms and point mutations 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.

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.

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 only minor alleles 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.

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.

In analyzing the significance of disease according to each analysis model, 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.

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 above, it was confirmed that the allele of the polymorphic sites of SEQ ID NOs. 1 to 5 did not affect the premature menopause.

In addition, it was confirmed from the results of Table 4 and Table 5 that the genotype of the polymorphic sites of SEQ ID NOs: 1 to 5 did not affect the onset of early menopause.

From the above results, it can be seen that the alleles and genotypes of the polymorphic regions of SEQ ID NOs: 1 to 5 are not statistically significant in early menopause diagnosis.

(3) haploid analysis

Table 6 and FIG. 1 show the association between the polymorphic sites for SEQ ID NOs 1 to 5, and FIG. 2 shows the haplotype formed from the Linkage Disequilibrium Block formed from FIG. 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 in the aforementioned FIG. 1 and Table 6 that the five 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 haplotype formed from an associative unbalanced block formed from FIG. As shown in FIG. 2, four haplotypes are shown from one of the associative non-equilibrium blocks. Among them, three haplotypes are analyzed except for haplotypes of which frequency is less than 0.1. It was observed to appear (Table 7). In addition, SEQ ID NOs: 2, 3, 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 patient and control groups. One haplotype was observed that showed a significant difference between the patient group and the normal group within one block formed. Haploid type was shown as AGTAC, which showed significance in haplotype, co-dominance, and dominant models, with a particularly high frequency in the control group. In conclusion, haplotype AGTAC is a haplotype that is specific to the control group. It can be predicted that a person with any of these haplotypes has resistance to early menopause.

Attach an electronic file to a sequence list

Claims (7)

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 haplotype determined from the polymorphic site of the DNA sequence of SEQ ID NOS: 1-5 from the nucleic acid sample, provided that the polymorphic sites of the DNA sequence of SEQ ID NOS: 1-5 are Single nucleotide polymorphism in a sample, comprising a 251 base, 201 base of SEQ ID NO: 2, 1117 base of SEQ ID NO: 3, 301 base of SEQ ID NO: 4, and 501 base of SEQ ID NO: 5) How to. The method of claim 1, wherein analyzing the haplotype
A sequence comprising a polymorphic site as a polynucleotide of SEQ ID NO: 1 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 251 base of the DNA sequence of SEQ ID NO: 1;
A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 2 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 201 base of the DNA sequence of SEQ ID NO: 2;
A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 3 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 1117th base of the DNA sequence of SEQ ID NO: 3;
A sequence comprising a polymorphic site as the polynucleotide of SEQ ID NO: 4 or a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 301th base of the DNA sequence of SEQ ID NO: 4; And
A polynucleotide of SEQ ID NO: 5 or a sequence comprising a polymorphic site as a complementary polynucleotide sequence thereof, wherein the polymorphic site refers to the 501th base of the DNA sequence of SEQ ID NO: 5
A method for detecting a single nucleotide polymorphism in a specimen, characterized in that it is carried out through sequencing of each polymorphic site using primers or probes each having a template. The method of claim 1, wherein analyzing the haplotype
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the nucleotide 251 (polymorphic site) is A, and the polynucleotide (a) consisting of 10 or more consecutive DNA sequences comprising the 251 base, or a complementary polynucleotide thereof ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) or a complementary polynucleotide consisting of 10 or more consecutive DNA sequences including the 201 base (G) and G comprising the 201 base ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 1117 th base (polymorphic site) is T, and the complementary polynucleotide thereof ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the polynucleotide (d) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the complementary polynucleotide thereof ; And
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, the 501th base (polymorphic site) is C and the polynucleotide (e) consisting of 10 or more consecutive DNA sequences comprising said 501th base, or a complementary polynucleotide thereof
Hybridizing the nucleic acid sample to a fixed microarray; And analyzing the obtained hybridization results to determine the nucleotide sequence of the polymorphic site. The method of claim 3, wherein the length of the DNA sequence of the polynucleotide or the complementary nucleotide thereof is 10 to 100 nucleotides in length in the microarray. In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the nucleotide 251 (polymorphic site) is A, and the polynucleotide (a) consisting of 10 or more consecutive DNA sequences comprising the 251 base, or a complementary polynucleotide thereof ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) or a complementary polynucleotide consisting of 10 or more consecutive DNA sequences including the 201 base (G) and G comprising the 201 base ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of 10 or more consecutive DNA sequences comprising the 1117 th base (polymorphic site) is T, and the complementary polynucleotide thereof ;
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the polynucleotide (d) consisting of ten or more consecutive DNA sequences comprising the 301th base (polymorphic site) is A and the complementary polynucleotide thereof ; And
In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, the 501th base (polymorphic site) is C and the polynucleotide (e) consisting of 10 or more consecutive DNA sequences comprising said 501th base, or a complementary polynucleotide thereof
A haplotype analysis kit determined from the polymorphic site of the DNA sequence of SEQ ID NOs: 1 to 5 for the early menopause diagnosis in Korean. The compound of claim 5, wherein the polynucleotide (a) or a complementary nucleotide thereof; Polynucleotide (b) or a complementary nucleotide thereof; Polynucleotide (c) or a complementary nucleotide thereof; Polynucleotide (d) or a complementary nucleotide thereof; And a polynucleotide (e) or a complementary nucleotide thereof is 10 to 100 nucleotides in length. The compound of claim 5, wherein the polynucleotide (a) or a complementary nucleotide thereof; Polynucleotide (b) or a complementary nucleotide thereof; Polynucleotide (c) or a complementary nucleotide thereof; Polynucleotide (d) or a complementary nucleotide thereof; And a microarray form each comprising a polynucleotide (e) or a complementary nucleotide thereof as a probe.

Images