KR101168737B1 - Polynucleotides derived from FANCA 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 or its complementary polynucleotide comprising a Single-Nucleotide Polymorphism (SNP) derived from a FANCA (Fanconi anemia) gene, wherein the polynucleotide or its complementary polynucleotide is premenopausal. As a marker for clinical diagnosis of (Premature Ovarian Failure; POF), it can be used.

Description

Polynucleotides derived from FANCA gene comprising 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 polymorph derived from the FANCA 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 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 FANCA (Fanconi anemia) gene. The present invention was completed.

Accordingly, an object of the present invention is to provide a polynucleotide or a complementary polynucleotide thereof comprising a monobasic polymorphism derived from the FANCA 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: 3, the polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 1809th base (polymorphic site) is A, and comprising the 1809th base ( a); In the polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, at least one from the group consisting of polynucleotide (b) consisting of 10 or more consecutive DNA sequences comprising the 201 base (C polymorphic site) and C; Selected polynucleotides 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 the polynucleotide of SEQ ID NO: 3 or 4 or its complementary polynucleotide from the nucleic acid sample, wherein the polymorphic site is the 1809th for the DNA sequence of SEQ ID NO: 3 A base, a DNA base of SEQ ID NO: 4 means the 201 base each) is provided, a method for detecting a single nucleotide polymorphism in a sample.

In addition, according to another aspect of the present invention, in order to provide information necessary for early menopause diagnosis, (i) obtaining a nucleic acid sample from a sample and (ii) polymorphism of the DNA sequence of SEQ ID NOS: 1-7 from the nucleic acid sample Site of the DNA sequence of SEQ ID NO: 1, 301 base for the DNA sequence of SEQ ID NO: 1, 201 base for the DNA sequence of SEQ ID NO: 2, 1809 base for the DNA sequence of SEQ ID NO: 3, and DNA sequence of SEQ ID NO: 4 For the DNA sequence of SEQ ID NO: 5, the 201 base for the DNA sequence of SEQ ID NO: 5, the 301 base for the DNA sequence of SEQ ID NO: 6, and the 401 base for the DNA sequence of SEQ ID NO: 7). A method is provided for detecting a monobasic polymorph in a specimen, characterized by an analysis of the body shape.

Polynucleotides (a) or (b), or their complementary polynucleotides, according to the present invention can be usefully used for the diagnosis of premature 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.

1 shows an associative non-equilibrium block between the monobasic polymorphs of SEQ ID NO: 1 to SEQ ID NO: 7.
Figure 2 shows a haplotype formed from the associative non-equilibrium block.

The present invention provides polynucleotides (a) or (b) derived from the sequence of SEQ ID NO: 3 or 4, or complementary polynucleotides thereof, newly found to be associated with premature menopause. In other words, the present invention provides a polynucleotide consisting of the DNA sequence of SEQ ID NO: 3, the polynucleotide (a) consisting of 10 or more consecutive DNA sequences comprising the 1809th base (polymorphic site) is A, the 1809th base; In the polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, at least one from the group consisting of polynucleotide (b) consisting of 10 or more consecutive DNA sequences comprising the 201 base (C polymorphic site) and C; Provided are selected polynucleotides or complementary polynucleotides thereof.

The DNA sequence of SEQ ID NO: 3 or 4 is a single nucleotide polymorphism with statistically significant difference (significance level p value <0.05) in the premenopausal patient group and the control group, and they are present in the FANCA gene located on chromosome 16. .

The present inventors conducted a variety of studies to select FANCA as a candidate gene associated with early menopause, 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 polymorphism of SEQ ID NO: 3 or 4, by the Hardy-Weinberg Equilibrium and logistic regression analysis, it was confirmed that there is a risk allele at each polymorphic site.

The present invention includes a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (a) and (b) or a complementary polynucleotide thereof, wherein the polynucleotide or the complementary polynucleotide thereof has a length of 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) and (b) according to the 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 at least one polynucleotide selected from the group consisting of the polynucleotides (a) and (b) 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. Preferably, the primer is aligned with the single nucleotide polymorphism of the 3'-end of SEQ ID NO: 3 or 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 processes, they can be used by other methods as well as GoldenGate Assay.

The present invention includes a premenopausal diagnostic probe and a premenopausal diagnostic microarray comprising the polynucleotide (a) and at least one selected from the group consisting of (b) 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) and (b) 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 the polynucleotide of SEQ ID NO: 3 or 4 or its complementary polynucleotide from the nucleic acid sample, wherein the polymorphic site is the 1809th for the DNA sequence of SEQ ID NO: 3 A base, a DNA base of SEQ ID NO: 4 means a 201 base, respectively); and a method for detecting a single nucleotide polymorphism in a 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). Then, 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 of SEQ ID NO: 3 or 4 or its complementary polynucleotide sequence, provided that the polymorphic site is In the case of the 1809th base, and the DNA sequence of SEQ ID NO: 4 means 201 base, respectively) can be carried out using a primer or probe as a template. The primer or probe is as described above.

In addition, analyzing the nucleotide sequence of the polymorphic site comprises hybridizing the nucleic acid sample to a microarray to which polynucleotide (a) or (b) or its complementary polynucleotide is immobilized, provided that the polynucleotide (a) Or (b) is 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.

The 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: 3 and 4 are A, C If one or more factors are present, it can be determined that they belong to a high 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 nucleotide sequence of the polymorphic site may comprise genotype (genotype) analysis of the polymorphic site of the SEQ ID NO: 3 or 4 DNA sequence. In other words, if the genotypes of the polymorphic regions of SEQ ID NOs: 3 and 4 contain at least one of A / A and A / G (SEQ ID NO: 3), and C / C and C / T (SEQ ID NO: 4), there is a risk of premature menopause. It can be judged as 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 includes a method for detecting a single nucleotide polymorphism in a sample comprising analyzing a haplotype from the nucleic acid sample to provide information necessary for early menopause diagnosis. Specifically, the haplotype analysis may be performed by analyzing a haplotype selected from the group consisting of haplotypes determined from the polymorphic sites of the DNA sequences of SEQ ID NOs: 1-7 from the nucleic acid sample. The polymorphic site of the DNA sequence of SEQ ID NO: 1 is 301 base, the DNA sequence of SEQ ID NO: 201 base, the DNA sequence of SEQ ID NO: 1809 base, the DNA sequence of SEQ ID NO: 4 201 base, the DNA sequence of SEQ ID NO: 5, the 201 base, the 301 base for the DNA sequence of SEQ ID NO: 6, and the 401 base for the DNA sequence of SEQ ID NO: 7, respectively.

That is, as a result of haplotype analysis, a person with an AGGTTGC haplotype of SEQ ID Nos. 1 to 7 can be determined to have a low risk of early menopause, and a person having an AGGCTGC haplotype has a high risk of early menopause. Can be determined (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 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 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)> 0.1, which are the criteria for polybasic polymorphism, were applied. Using the frequency of appearance between patient and control group, each monobasic polymorphism was analyzed with significance level <0.05 and analyzed using SAS software version 9.1.3. 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 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.

5. Results

(1) monobasic polymorphisms showing significance in the FANCA gene

Table 3 shows the results of analyzing the association between the control group and the patient group and early menopause. Among the polynucleotides of SEQ ID NOs: 1 to 7, polymorphic sequences of SEQ ID NOs: 3 and 4 show statistically significant differences. That was analyzed.

SEQ ID NO: Reference
SNP ID Allele location SNP Role Amino acid changes One rs9282681 A> G 88333415 missense Thr → Ala 2 rs3743860 G> A 88345992 intron No change 3 rs1006547 G> A 88371730 intron No change 4 rs2239359 T> C 88376981 missense Gly → Ser 5 rs2239360 T> C 88377084 intron No change 6 rs11646374 G> A 88385436 missense Ala → Val 7 rs6500452 C> T 88386006 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: 1-7 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 and their genotypes 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 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 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 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 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 conducted in three categories: the allele model, the co-dominant model, the dominant genotype, and the recessive model. The meaning of each column in Table 6 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. 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 significance of the difference between the control and patient groups by 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.

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 each of the three analytical models, if the majority allele is A1 and the minority 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.

Alleles can be used to determine the degree of association (risk) of a risk allele with a 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 can be seen from Table 5, at least one nucleotide sequence of the SEQ ID NO: 3 and 4 polymorphic sites is A, C (risk allele), respectively If present, the risk of premature menopause is high.

In addition, the similarity and dominance with the SEQ ID NOs 3 and 4 alleles Significant in the model, SEQ ID NOs: 3 and 4 show that the odds ratio is> 1, and those with genotype Minor Allele tend to premature menopause than those with Major Allele. The risk was higher, and those who had at least one minor allele had a higher risk of premature menopause than those with two algebraic alleles.

From the above results, the genotype of the polymorphic regions of SEQ ID NOs: 3 and 4 is premature menopause when at least one of A / A and A / G (SEQ ID NO: 3) and C / C and C / T (SEQ ID NO: 4) is present. It can be judged that the risk is high, and if one or more nucleotide sequences of the polymorphic regions of SEQ ID NOs: 3 and 4 are A and C (risk alleles), respectively, the risk of early menopause can be determined. have. 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 polymorphism of SEQ ID NO: 3 or 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 6 and FIG. 1 show the association between the polymorphic sites for SEQ ID NOs: 1-7, and FIG. 2 shows a haplotype formed in comparison with the results of genotyping from the Linkage Disequilibrium Block formed from FIG. Indicates. Table 8 shows the results of the association test with early menopause for haplotype of each block.

Table 6 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 base polymorphism shown in Fig. 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, single nucleotide polymorphisms of SEQ ID NOs: 1 to 7 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 in comparison to genotyping results from the associative non equilibrium block formed from FIG. The haplotype corresponding to FIG. 2 appears from each of the unrelated equilibrium blocks shown in FIG. 1, and the haplotypes appearing more frequently in the patient group and the haplotypes appearing in the normal group were observed. Table 7). 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 is a table examining the significant difference in the frequency of the haplotypes of Figure 2 between the patient group and the normal group.

Two haplotypes were observed that showed significant differences between the patient and normal groups within the two blocks formed. In the first block, ht1 appears as A-G-G-T-T-G-C, which is unusually high in the normal group. Haploid ht2, on the other hand, appears as A-G-G-C-T-G-C, which is a high frequency specific to the patient group. In conclusion, in block 1, ht1 is a haplotype that is specific in the normal group, and it can be predicted that those who have this haplotype are resistant to premature menopause. People with body types can be expected to be sensitive 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.

Attach an electronic file to a sequence list

Claims (12)

delete delete In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the 201 base (polymorphic site) is C and consists of a polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 201 base or a complementary polynucleotide thereof Amplification primers for menopause diagnosis. In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the 201 base (polymorphic site) is C and consists of a polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 201 base or a complementary polynucleotide thereof Menopause diagnostic probe. Microarray comprising a premenopausal diagnostic probe according to claim 4. In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, the 201 base (polymorphic site) is C, and comprises a polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 201 base or a complementary polynucleotide thereof, Early Menopause Diagnostic Kit. 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 the polynucleotide of SEQ ID NO: 4 or its complementary polynucleotide from the nucleic acid sample, wherein the polymorphic site means the 201 base of the DNA sequence of SEQ ID NO: 4 A method for detecting a monobasic polymorphism in a sample. 8. The method of claim 7, wherein analyzing the nucleotide sequence of the polymorphic site comprises a polynucleotide of SEQ ID NO: 4 or a complementary polynucleotide sequence thereof comprising the polymorphic site, wherein the polymorphic site is a DNA sequence of SEQ ID NO: Means a 201 base of;) using a primer or a probe as a template, a method for detecting a single nucleotide polymorphism in a sample. The method of claim 7, wherein the analyzing the base sequence of the polymorphic site comprises at least 10 or more polynucleotides consisting of the DNA sequence of SEQ ID NO: 4, wherein the 201th base (polymorphic site) is C and the 201th base is included. Hybridizing the nucleic acid sample to a micronucleotide consisting of a contiguous DNA sequence or a complementary polynucleotide thereof; And analyzing the obtained hybridization result. 10. The method of claim 9, wherein the DNA sequence of the polynucleotide immobilized on the microarray or its complementary polynucleotide is 10 to 100 nucleotides in length. The method according to claim 7, wherein the step of analyzing the nucleotide sequence of the polymorphic site is a genotype of a polymorphic site of the DNA sequence of SEQ ID NO: 4, wherein the polymorphic site means the 201 base of the DNA sequence of SEQ ID NO: 4 genotype), characterized in that the detection of a single nucleotide polymorphism in the sample. delete

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