KR102060653B1 - Analytical method for diagnosing premature ovarian failure and kit therefor - Google Patents

Abstract

In order to provide information necessary for diagnosing premenopausal premature menopause, in particular, premature menopause caused by environmental hazards, the expression level of the protein coding for USP49, i.e. It provides an analysis method comprising the step of measuring. The present invention also provides a kit for diagnosing early menopause, including early menopause caused by formaldehyde, which is an environmental harmful factor including a molecule capable of measuring the expression level of a gene encoding the protein of SEQ ID NO: 1 or 2.

Description

Analytical method for diagnosing premature ovarian failure and kit therefor}

The present invention relates to an analytical method and kit for diagnosing early menopause for providing information necessary for early menopause diagnosis.

Women experience menopause after age 50 (De Bruin, J.P., et al., The role of genetic factors in age at natural menopause. Hum Reprod, 2001. 16 (9): p. 2014-8). However, premature ovarian failure is a pathological condition in which menopause occurs before the age of forty (Jankowska, K., Premature ovarian failure.Prz Menopauzalny, 2017.16 (2)). Premenopausal dysmenorrhea for more than 6 months before age 40 and blood follicle stimulating hormone> 40 mlU / ml is diagnosed as early menopause (Rebar, RW, GF Erickson, and SS Yen, Idiopathic premature ovarian failure: clinical and endocrine characteristics. Steril, 1982. 37 (1)).

The causes of early menopause are known as infections, surgery, chemotherapy or radiation therapy, ovarian damage, autoimmune diseases, genetic diseases, but the exact cause is still unclear. For example, formaldehyde, one of the environmentally harmful factors that can easily be encountered around us, is known to cause early menopause, but the exact mechanism of development is not yet known.

Ubiquitin binds to the target protein and acts as a marker for the proteasome to recognize. This process is called ubiquitination, and proteasomes break down proteins that bind ubiquitin. This process is called deubiquitination (Deubiquitination) process that inhibits the decomposition of proteins. Deubiquitinating enzymes, also referred to as proteolytic enzymes, cleave the ubiquitin attached to the target protein, preventing the protein from being recognized and degraded by the proteasome. There are about 100 deubiquitination enzymes in the body, and they increase the stability of the protein, so if there is an abnormal expression, the protein does not perform a specific function. Recently, the present inventors have reported the results of screening proteolytic enzyme genes abnormally expressed in early menopause through multiplex PCR (Kim, SY, et al., Ubiquitin-specific peptidase). 5 and ovarian tumor deubiquitinase 6A are differentially expressed in p53 + / + and p53 - / -. HCT116 cells Int J Oncol, 2018).

The present inventors constructed an early menopause model by exposing formaldehyde, an environmentally harmful factor that causes premature menopause, to ovarian cell lines, and then searched for proteolytic enzyme genes that express expression differences through multiple polymerase chain reactions. . As a result, it was found that the expression level of USP49, one of the protease regulating enzymes, increased as the amount of exposed formaldehyde increased. Therefore, detection of USP49 overexpression can be usefully used to diagnose early menopause, and USP49 can be used as a biomarker for early menopause diagnosis.

Accordingly, an object of the present invention is to provide an analysis method using USP49, in order to provide information necessary for early menopause diagnosis.

In addition, an object of the present invention is to provide a kit for diagnosing early menopause comprising a molecule capable of measuring the expression level of the gene encoding the USP49.

According to one aspect of the present invention, in order to provide information necessary for early menopause diagnosis, an analysis method is provided that includes measuring the expression level of a gene encoding a protein of SEQ ID NO: 1 or 2 in a subject's sample.

In the assay method of the present invention, the early menopause may be early menopause caused by formaldehyde. In addition, in the analysis method of the present invention, the subject sample may be blood or urine. Determination of the expression level of the gene may be performed by measuring the amount of mRNA or protein. In one embodiment, the measurement of the amount of protein can be performed by western blotting. In another embodiment, the measurement of the mRNA amount can be performed via RT-PCR or real-time-PCR.

According to another aspect of the present invention, a kit for diagnosing early menopause comprising a molecule capable of measuring the expression level of a gene encoding a protein selected from the group consisting of the protein of SEQ ID NO: 1 or 2, wherein the molecule is specific for the protein. Antibodies, substrates, ligands, or cofactors that bind to proteins; Alternatively, there is provided a kit for early menopause diagnostic, which is a primer having a complementary sequence specific for the gene encoding the protein.

In the diagnostic kit of the present invention, the early menopause may be early menopause caused by formaldehyde. In one embodiment, the molecule can be labeled with a detectable label. In another embodiment, the primer may be in the form of a microarray immobilized on a substrate. In addition, the primer may have a base sequence selected from the base sequences of SEQ ID NOs: 5 to 8.

It has been found by the present invention that the amount of expression in the ovarian cell line of USP49, one of the protease regulating enzymes, increases with increasing amount of exposed formaldehyde. Thus, the assays and kits according to the invention can be usefully used to diagnose premature menopause, particularly premature menopause caused by formaldehyde.

Figure 1 shows the result of measuring the expression of proteolytic regulatory enzymes according to the increase in formaldehyde in OVCAR5 cells, ovary-derived cell line through Multiplex PCR (A: 125 μM, B: 250 μM, C: 500 μM, D: 1000 μM, formaldehyde treatment concentration)
Figure 2 shows the results of measuring the expression of proteolytic enzymes in accordance with the increase in formaldehyde in A2780 cells, ovarian-derived cell line by Multiplex PCR (A: 125 μM, B: 250 μM, C: 500 μM, D: 1000 μM, formaldehyde treatment concentration).
Figure 3 shows the results of statistical analysis of the relative expression ratio of the proteolytic regulatory enzyme USP49 based on the results of Figure 1.
Figure 4 shows the results of statistical analysis of the relative expression ratio of the proteolytic regulatory enzyme USP49 based on the results of Figure 2.
Figure 5 shows the results of measuring the relative expression level of USP49 by performing qRT-PCR.

The present inventors exposed the formaldehyde causing early menopause to ovarian cell lines by concentration (125 μM, 250 μM, 500 μM, 1000 μM) to build an early menopause model, and then extracted RNA to synthesize cDNA. Multiplex PCR (Multiplex PCR) was used to confirm the gene expression level of proteolytic enzymes in cell lines exposed to formaldehyde. I checked. Three or more repeated experiments confirmed that the expression level increased as the amount of formaldehyde exposed to the protease regulatory enzyme USP49 was increased in two ovary-derived cell lines A2780 and OVCAR5. Therefore, the detection of overexpression of the proteolytic enzyme USP49 in the subject's sample may be useful for diagnosing premenopause, especially premenopausal caused by formaldehyde, an environmentally harmful factor. Menopause, can be used as a biomarker for the diagnosis of early menopause caused by formaldehyde.

The present invention provides an analysis method comprising the step of measuring the expression level of the gene encoding the USP49 protein in a sample of the subject, in order to provide information necessary for early menopause diagnosis. In the assay method of the present invention, the early menopause may be early menopause caused by formaldehyde.

Human USP49 has been identified with two isoforms, each having the sequence of the protein of SEQ ID NO: 1 or 2. Thus, unless otherwise indicated herein, USP49 comprises a protein of SEQ ID NO: 1 or 2, preferably a protein of SEQ ID NO: 1. In one embodiment, the gene encoding the USP49 protein is a gene encoding the protein of SEQ ID NO: 1.

In the analysis method of the present invention, the subject's sample refers to a sample separated from the human body in vitro, and includes, for example, blood, urine, etc. separated from the human body in vitro.

The assay method of the present invention comprises measuring the expression level of the gene encoding USP49, ie, the protein of SEQ ID NO: 1 or 2. The gene sequence encoding the USP49 may be a known gene (for example, a gene selected from the group consisting of nucleotide sequences of SEQ ID NO: 3 or 4) known in the gene bank.

Determination of the expression level of the gene can be performed by measuring the level of the mRNA or protein (ie, the protein of SEQ ID NO: 1 or 2) of the gene according to the method commonly used in the field of biotechnology. The measurement of USP49 protein amount can be measured by a method such as western blotting. In the case of measuring the amount of protein by Western blotting method, if the measured protein expression level is significantly higher than that of a normal person, it can be determined as a premenopausal risk patient. In addition, the mRNA amount of the gene encoding the USP49 protein (for example, a gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 4) may be measured by reverse transcription-PCR or real-time PCR. It can measure by a method.

The present invention also provides a kit for diagnosing early menopause comprising a molecule capable of measuring the expression level of USP49, ie, a protein of SEQ ID NO: 1 or 2, preferably a protein of SEQ ID NO: 1, wherein the molecule is the protein Antibodies, substrates, ligands, or cofactors that specifically bind to; Or it provides a kit for early menopause diagnostics which is a primer having a complementary sequence specific for the gene encoding the protein. In the diagnostic kit of the present invention, the early menopause may be early menopause caused by formaldehyde.

Examples of molecules capable of measuring the expression level of a gene encoding USP49 include antibodies, substrates, ligands, or cofactors that specifically bind to the protein; Or a primer having a complementary sequence specific for the gene encoding the protein.

USP49 protein can produce a polyclonal antibody or monoclonal antibody according to methods commonly used in the field of biotechnology, and can prepare a diagnostic kit comprising the antibody. In addition, since USP49 has been shown to function as a deubiquitination enzyme, the kit of the present invention may be prepared to include a substrate, a ligand, or a cofactor. In addition, a primer having a complementary sequence specific for a gene encoding USP49 may be prepared according to a method commonly used in the field of biotechnology, and a diagnostic kit including the primer may be prepared. In one embodiment, the primer is a forward primer: 5'-AGG ACT ACG TGC TCA ATG ATA ACC-3 '(SEQ ID NO: 5) and / or reverse primer: 5'-GCA GGA GCA GCC GTG CAC TCT-3' ( SEQ ID NO: 6). In other embodiments, the primer is a forward primer: 5'-CCC TGA ACG CTA TCA CTG CA-3 '(SEQ ID NO: 7) and / or reverse primer: 5'-TTG GCC AGC ATC TCA GTG AG-3' Number 8).

In the diagnostic kit of the present invention, a molecule capable of measuring the expression level of a gene encoding USP49 protein may be labeled with a detectable label (eg, a chromophore, etc.). In addition, the diagnostic kit of the present invention may be in the form of a chip such as a DNA chip or a protein chip by having a microarray in which the primer is immobilized on a substrate.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

Example

1. Test Method

(1) Cell culture and sample preparation

Ovarian-derived cell line OVCAR5 (TCP-1021, ATCC, Manassas, VA, USA) is Dulbecco's modified Eagle's medium containing 10% FBS, 1% penicillin / streptomycin (Gibco, Grand Island, NY, USA) (DMEM, Gibco, Grand Island, NY, USA). Ovarian derived cell line A2780 (CRL-2722, ATCC, Manassas, VA, USA)) contains RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% FBS, 1% Antibiotic-Antimycotic (Gibco, Grand Island, NY, USA) , USA). Both cell lines were incubated at 37 ° C. in a 5% CO ₂ incubator. Formaldehyde was treated at ovarian derived cell lines OVCAR5 and A2780 at concentrations of 125 μM, 250 μM, 500 μM, 1000 μM and harvested after 12 hours.

(2) RNA extraction and cDNA synthesis

RNA was extracted using Trizol according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). After cDNA was synthesized using cDNA synthesis kit (Cosmogenetech, Seoul, Korea) with 2 μg concentration of RNA, the samples were diluted 1/10 for Multiplex PCR.

(3) Multiplex PCR

PCR is performed using primers that can amplify the housekeeping gene GAPDH on each cDNA diluted to the same concentration. Multiplex PCR is performed when the amount of GAPDH amplified is constant. Multiplex PCR was performed by adding 2X premix (SMP01-M25h; Solgent, Daejeon, South Korea) primer for Multiplex PCR to each cDNA. PCR conditions were performed 40 times in 20 seconds at the denaturation step 95 ℃, 40 seconds at the binding step 60 ℃, 60 seconds at 72 ℃ extension stage. Primer sequences used are shown in Tables 1 to 3 below.

(4) Check and analyze the results

Multiplex PCR products were electrophoresed on a 2% agarose gel. The bands amplified in cDNA by the respective primers were identified through the gel Doc, and proteolytic enzymes showing a tendency to decrease or increase according to formaldehyde concentration were analyzed.

(5) qRT-PCR

QPR- using USP49 primer (forward: 5'-CCC TGA ACG CTA TCA CTG CA-3 '(SEQ ID NO: 7), reverse: 5'-TTG GCC AGC ATC TCA GTG AG-3' (SEQ ID NO: 8)) PCR was performed. The cDNA was amplified using the SyBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc), and relative expression levels were analyzed by normalizing ^GADPH with controls and then analyzed by the 2- ^ΔΔCq method.

2. Test result

(1) Analysis of Proteolytic Enzyme Gene Expressed by Multiplex PCR

RNA was extracted from ovarian-derived cell lines (OVCAR5 and A2780) treated with formaldehyde concentration, cNDA was synthesized, and then mixed with 12 groups of primers and cDNA, followed by multiplex PCR, followed by gel electrophoresis. It was. 1 and 2 are the results of measuring the expression of proteolytic enzymes according to formaldehyde increase in OVCAR5 cells (FIG. 1) and A2780 cells (FIG. 2), which are ovarian-derived cell lines, using Multiplex PCR, respectively (A: 125 μM). , B: 250 μM, C: 500 μM, D: 1000 μM, formaldehyde treatment concentration). The results were repeated three times. It was confirmed that USP49 was a protease regulating enzyme whose expression was increased due to the increase in formaldehyde concentration in both OVCAR5 cells and A2780 cells. Based on the results of FIGS. 1 and 2, the results of statistical analysis of the relative expression ratio of the protease regulatory enzyme USP49 are shown in FIGS. 3 (OVCAR5 cells) and 4 (A2780 cells), respectively.

In addition, the result of measuring the relative expression level by performing qRT-PCR to quantitatively analyze the expression of USP49 affected by formaldehyde is shown in FIG. From the results of FIG. 5, it can be seen that as the formaldehyde treatment concentration increases, USP49 mRNA levels increase.

3. Consideration

Formaldehyde is an environmental hazard that can easily be encountered around us and is known as one of the premature menopauses. The present inventors confirmed that the protein-regulating enzyme gene USP49 is increased in a concentration-dependent manner in a formaldehyde-treated ovary-derived cell line through Multiplex PCR. Therefore, the early menopausal mechanism model by formaldehyde, which is an environmental harmful factor, can be built around the protease-regulating enzyme gene USP49. In addition, according to the results of this study, it is possible to study the mechanism of early menopause caused by formaldehyde, an environmentally harmful factor, and use the proteolytic enzyme gene USP49 as a biomarker for early menopause diagnosis caused by formaldehyde.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Analytical method for diagnosing premature ovarian failure and          kit therefor <130> PN0855 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 688 <212> PRT <213> Homo sapiens <400> 1 Met Asp Arg Cys Lys His Val Gly Arg Leu Arg Leu Ala Gln Asp His   1 5 10 15 Ser Ile Leu Asn Pro Gln Lys Trp Cys Cys Leu Glu Cys Ala Thr Thr              20 25 30 Glu Ser Val Trp Ala Cys Leu Lys Cys Ser His Val Ala Cys Gly Arg          35 40 45 Tyr Ile Glu Asp His Ala Leu Lys His Phe Glu Glu Thr Gly His Pro      50 55 60 Leu Ala Met Glu Val Arg Asp Leu Tyr Val Phe Cys Tyr Leu Cys Lys  65 70 75 80 Asp Tyr Val Leu Asn Asp Asn Pro Glu Gly Asp Leu Lys Leu Leu Arg                  85 90 95 Ser Ser Leu Leu Ala Val Arg Gly Gln Lys Gln Asp Thr Pro Val Arg             100 105 110 Arg Gly Arg Thr Leu Arg Ser Met Ala Ser Gly Glu Asp Val Val Leu         115 120 125 Pro Gln Arg Ala Pro Gln Gly Gln Pro Gln Met Leu Thr Ala Leu Trp     130 135 140 Tyr Arg Arg Gln Arg Leu Leu Ala Arg Thr Leu Arg Leu Trp Phe Glu 145 150 155 160 Lys Ser Ser Arg Gly Gln Ala Lys Leu Glu Gln Arg Arg Gln Glu Glu                 165 170 175 Ala Leu Glu Arg Lys Lys Glu Glu Ala Arg Arg Arg Arg Arg Glu Val             180 185 190 Lys Arg Arg Leu Leu Glu Glu Leu Ala Ser Thr Pro Pro Arg Lys Ser         195 200 205 Ala Arg Leu Leu Leu His Thr Pro Arg Asp Ala Gly Pro Ala Ala Ser     210 215 220 Arg Pro Ala Ala Leu Pro Thr Ser Arg Arg Val Pro Ala Ala Thr Leu 225 230 235 240 Lys Leu Arg Arg Gln Pro Ala Met Ala Pro Gly Val Thr Gly Leu Arg                 245 250 255 Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Ile Leu Gln Val Leu Ser             260 265 270 His Leu Gln Lys Phe Arg Glu Cys Phe Leu Asn Leu Asp Pro Ser Lys         275 280 285 Thr Glu His Leu Phe Pro Lys Ala Thr Asn Gly Lys Thr Gln Leu Ser     290 295 300 Gly Lys Pro Thr Asn Ser Ser Ala Thr Glu Leu Ser Leu Arg Asn Asp 305 310 315 320 Arg Ala Glu Ala Cys Glu Arg Glu Gly Phe Cys Trp Asn Gly Arg Ala                 325 330 335 Ser Ile Ser Arg Ser Leu Glu Leu Ile Gln Asn Lys Glu Pro Ser Ser             340 345 350 Lys His Ile Ser Leu Cys Arg Glu Leu His Thr Leu Phe Arg Val Met         355 360 365 Trp Ser Gly Lys Trp Ala Leu Val Ser Pro Phe Ala Met Leu His Ser     370 375 380 Val Trp Ser Leu Ile Pro Ala Phe Arg Gly Tyr Asp Gln Gln Asp Ala 385 390 395 400 Gln Glu Phe Leu Cys Glu Leu Leu His Lys Val Gln Gln Glu Leu Glu                 405 410 415 Ser Glu Gly Thr Thr Arg Arg Ile Leu Ile Pro Phe Ser Gln Arg Lys             420 425 430 Leu Thr Lys Gln Val Leu Lys Val Val Asn Thr Ile Phe His Gly Gln         435 440 445 Leu Leu Ser Gln Val Thr Cys Ile Ser Cys Asn Tyr Lys Ser Asn Thr     450 455 460 Ile Glu Pro Phe Trp Asp Leu Ser Leu Glu Phe Pro Glu Arg Tyr His 465 470 475 480 Cys Ile Glu Lys Gly Phe Val Pro Leu Asn Gln Thr Glu Cys Leu Leu                 485 490 495 Thr Glu Met Leu Ala Lys Phe Thr Glu Thr Glu Ala Leu Glu Gly Arg             500 505 510 Ile Tyr Ala Cys Asp Gln Cys Asn Ser Lys Arg Arg Lys Ser Asn Pro         515 520 525 Lys Pro Leu Val Leu Ser Glu Ala Arg Lys Gln Leu Met Ile Tyr Arg     530 535 540 Leu Pro Gln Val Leu Arg Leu His Leu Lys Arg Phe Arg Trp Ser Gly 545 550 555 560 Arg Asn His Arg Glu Lys Ile Gly Val His Val Val Phe Asp Gln Val                 565 570 575 Leu Thr Met Glu Pro Tyr Cys Cys Arg Asp Met Leu Ser Ser Leu Asp             580 585 590 Lys Glu Thr Phe Ala Tyr Asp Leu Ser Ala Val Val Met His His Gly         595 600 605 Lys Gly Phe Gly Ser Gly His Tyr Thr Ala Tyr Cys Tyr Asn Thr Glu     610 615 620 Gly Gly Phe Trp Val His Cys Asn Asp Ser Lys Leu Asn Val Cys Ser 625 630 635 640 Val Glu Glu Val Cys Lys Thr Gln Ala Tyr Ile Leu Phe Tyr Thr Gln                 645 650 655 Arg Thr Val Gln Gly Asn Ala Arg Ile Ser Glu Thr His Leu Gln Ala             660 665 670 Gln Val Gln Ser Ser Asn Asn Asp Glu Gly Arg Pro Gln Thr Phe Ser         675 680 685 <210> 2 <211> 640 <212> PRT <213> Homo sapiens <400> 2 Met Asp Arg Cys Lys His Val Gly Arg Leu Arg Leu Ala Gln Asp His   1 5 10 15 Ser Ile Leu Asn Pro Gln Lys Trp Cys Cys Leu Glu Cys Ala Thr Thr              20 25 30 Glu Ser Val Trp Ala Cys Leu Lys Cys Ser His Val Ala Cys Gly Arg          35 40 45 Tyr Ile Glu Asp His Ala Leu Lys His Phe Glu Glu Thr Gly His Pro      50 55 60 Leu Ala Met Glu Val Arg Asp Leu Tyr Val Phe Cys Tyr Leu Cys Lys  65 70 75 80 Asp Tyr Val Leu Asn Asp Asn Pro Glu Gly Asp Leu Lys Leu Leu Arg                  85 90 95 Ser Ser Leu Leu Ala Val Arg Gly Gln Lys Gln Asp Thr Pro Val Arg             100 105 110 Arg Gly Arg Thr Leu Arg Ser Met Ala Ser Gly Glu Asp Val Val Leu         115 120 125 Pro Gln Arg Ala Pro Gln Gly Gln Pro Gln Met Leu Thr Ala Leu Trp     130 135 140 Tyr Arg Arg Gln Arg Leu Leu Ala Arg Thr Leu Arg Leu Trp Phe Glu 145 150 155 160 Lys Ser Ser Arg Gly Gln Ala Lys Leu Glu Gln Arg Arg Gln Glu Glu                 165 170 175 Ala Leu Glu Arg Lys Lys Glu Glu Ala Arg Arg Arg Arg Arg Glu Val             180 185 190 Lys Arg Arg Leu Leu Glu Glu Leu Ala Ser Thr Pro Pro Arg Lys Ser         195 200 205 Ala Arg Leu Leu Leu His Thr Pro Arg Asp Ala Gly Pro Ala Ala Ser     210 215 220 Arg Pro Ala Ala Leu Pro Thr Ser Arg Arg Val Pro Ala Ala Thr Leu 225 230 235 240 Lys Leu Arg Arg Gln Pro Ala Met Ala Pro Gly Val Thr Gly Leu Arg                 245 250 255 Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Ile Leu Gln Val Leu Ser             260 265 270 His Leu Gln Lys Phe Arg Glu Cys Phe Leu Asn Leu Asp Pro Ser Lys         275 280 285 Thr Glu His Leu Phe Pro Lys Ala Thr Asn Gly Lys Thr Gln Leu Ser     290 295 300 Gly Lys Pro Thr Asn Ser Ser Ala Thr Glu Leu Ser Leu Arg Asn Asp 305 310 315 320 Arg Ala Glu Ala Cys Glu Arg Glu Gly Phe Cys Trp Asn Gly Arg Ala                 325 330 335 Ser Ile Ser Arg Ser Leu Glu Leu Ile Gln Asn Lys Glu Pro Ser Ser             340 345 350 Lys His Ile Ser Leu Cys Arg Glu Leu His Thr Leu Phe Arg Val Met         355 360 365 Trp Ser Gly Lys Trp Ala Leu Val Ser Pro Phe Ala Met Leu His Ser     370 375 380 Val Trp Ser Leu Ile Pro 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Thr Met Glu Pro Tyr Cys Cys Arg Asp Met Leu Ser Ser Leu Asp             580 585 590 Lys Glu Thr Phe Ala Tyr Asp Leu Ser Ala Val Val Met His His Gly         595 600 605 Lys Gly Phe Gly Ser Gly His Tyr Thr Ala Tyr Cys Tyr Asn Thr Glu     610 615 620 Gly Gly Ala Cys Ala Leu Leu Cys Gly Val Gly Asp Thr Glu Arg Gly 625 630 635 640 <210> 3 <211> 8822 <212> DNA <213> Homo sapiens <400> 3 gtaatagaga agaggaaacc taaccaatga ctgtggaata atatggaaga agatggaaaa 60 aacttgttta atgggttgag aaagtggcga cttctataag acatggatag atgcaaacat 120 gtagggcggt tacggctcgc ccaggaccac tccatcctga accctcagaa gtggtgctgc 180 ttagagtgtg ccaccaccga gtccgtgtgg gcctgcctca agtgctccca cgtggcctgc 240 ggccgctata ttgaggacca cgccctgaaa cactttgagg agacgggaca cccgctagcc 300 atggaagtcc gggatctcta cgtgttctgt tacctgtgca aggactacgt gctcaatgat 360 aacccagagg gggacctgaa gctgctaaga agctccctcc tggcggtccg gggccagaaa 420 caggacacgc cggtgagacg tgggcggacg ctgcggtcca tggcttcggg tgaggacgtg 480 gtcctgccgc agcgcgctcc tcagggacag ccgcagatgc tcacggctct 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1380 cccttctccc agaggaagct caccaaacag gtcttaaagg tggtgaatac catatttcat 1440 gggcagctgc tcagtcaggt cacatgtata tcatgcaatt acaaatccaa taccattgag 1500 cccttttggg acctatccct ggaattccct gaacgctatc actgcataga aaaggggttt 1560 gtccctttga atcaaacaga gtgcttgctc actgagatgc tggccaaatt cacagagaca 1620 gaggccctgg aagggagaat ctacgcttgt gaccagtgta acagcaaacg acgaaaatcc 1680 aatcccaaac cccttgttct gagtgaagct agaaagcagt taatgatcta cagactacct 1740 caggttctcc ggctgcacct taaaagattc aggtggtctg gccgtaatca tcgagagaag 1800 attggggtcc atgtcgtctt tgaccaggta ttaaccatgg aaccttactg ctgcagggac 1860 atgctctcct ctcttgacaa agagaccttt gcctatgatc tctccgcagt ggtcatgcat 1920 cacgggaaag ggtttggctc aggacactac acagcctatt gctacaacac agagggaggt 1980 ttttgggtcc actgcaatga ctcaaagctg aatgtatgca gtgtcgagga agtgtgcaaa 2040 acccaggcct acatcctttt ttacactcaa agaacagtgc agggcaatgc aagaatctca 2100 gaaacccatc tccaagctca ggtgcagtcc agcaacaatg atgaaggcag accacagaca 2160 ttttcctgaa tgggaggcat gtatcaaaga ctggcttttg tgtattggtg tccatacatc 2220 tttcctctta taggaaataa ggctactgca ggaacagatt actaggtttg ccttgctggg 2280 tctagagtag aaggtgccag gtgattcgtg tcctgtcgta aattttatag tctctttctt 2340 ttgaatgagt ttgggagttc cctcctttcc taaaaacaaa agaagtaact tctatgaaga 2400 tcctttcatc agttgcttct gaatatagag ggatctgggt ggaggaaagc ggggcggggg 2460 ggtaaatcaa tcatatccac acaggagagc agccatgttt tctctccatg atgtgattcc 2520 cctgctaatc aggacctcct tgcagcacca gaagaaccct ccggatgtga gcagtcctgg 2580 acagagcacg tgaggagggg ccctgggcta gcagtttgct ggatgggatt ggctccaaga 2640 tgggagatga agtctaaagg gtttattccc acaaaggaag taaccttcaa gggtttaaaa 2700 caaacttcct tttttgggga tagcagctga agagtggagt gagatgcatg gaacaaattt 2760 ccccatgcaa agtgggatgg ttttcatatg tcataccctc cctacttgct gctgtcatat 2820 aatcattcca tacttctgag actggcatga ataccccttc attctaaaga aggggcaggt 2880 catataaaga agcaatagac tagcacatgt tccatgctga gccaaaggac actcctggct 2940 atctcagttc tgtagcagat ccctggttca ggtctagggc aagcacatgg tttggtttaa 3000 attgattctt aggttttctc agaactcaat tgagggtggg cagagatagc tgatgctcca 3060 gggcttattg aaggcctgtc acccgttctc acttcagtgc ggcatgcagc ttcttagcct 3120 gcagcttctt tcttgctcct ggagagctag aacattcatg ttcacagggt ttgggagctt 3180 ttggttctct agggtatgtt aactgcttag gagtccacct cgtacatatc tgaagagttg 3240 attaccaatg gccttgacct gtttcctccc aaatctgata atccctactg ttgctttacc 3300 ccccatactc agtccatcag agaaaaaaac aagagtgccc cgtcctttcc cccagtgcta 3360 gtaaaataca tcttcctttt taaattgggt cagtttctaa acagaatgaa attgcccaga 3420 taaaaaccct tgtccccatc aggcaccact tcattcttag ttcttcactt agatttgctg 3480 gtgaaagtga atgcaaaccc taacttttgt tgctgtggtc actgcagtga gcctcactca 3540 acctgtcact atgaatgctc atcttttgac tgagtatatc atattaatgt ttgaattcag 3600 gtagaagtac agttgtcact tgaagggagt agatgtggaa gggcgttcgg ggaggagtgg 3660 gaagtgggga aagccaggct aaagaatgag ggttatagct ttagcaatgt tgatgagcca 3720 catttcccac acagatctgt agcatttgct gttgacctct tttgagatga actctcccat 3780 aggccctgcc aggcagcact gagacatgga ggaaattcag acaaaaaatg gctcaccctg 3840 atagggaagg agtgactata ttttaaatta aacttgcttt ttccaacact gtgaggtttc 3900 tgtaatattt agcttttatt tggaagcgat agcgtatggc attttttatg ctgtttggtt 3960 tatattgtct actgcaggct tctttgtata agctttgcct gggctcaccc tctcctggac 4020 actgttttaa agtgtcaccg ctgtccatgc tgccagaggc tctgatgtga agctgtactc 4080 tcaagatcca cgtttttaag tcggcatgat tagtaatgac tttttgtggc aatagaaaag 4140 cccattaact caaaaaaacc aaggagagta aaaaggaaag gaaactatga atcgtgcctc 4200 atctttcatt gaatcctata tttattattc tagaaaaaaa gaacttacaa gatagagaag 4260 aactaataga gcatctactt tacccctcag gctgtccagt ggggataatt attaataaac 4320 atttaatgga aaattcaaag atcttccaat ttaattttta aaaatagttt tataccaaat 4380 attataagga tttattaagc agattaagga ctttgtgaaa taagtgctta acttagctga 4440 gcacgcatat atcaaagtaa taaaagaaac taacatcaga gtctccactg agctacacat 4500 ttgtgaaagg tgcccactgc agttacagac acctcaggta cccagaggcg gcagcactgg 4560 acacttagga cgttagtatt taaggagatt tttattaaag aagagttccc tgctgcataa 4620 gccaatggct attgtgaggg aaatgtccct ttgcctttaa tttatttacg tactgggtat 4680 agatgaatgt ctcagttgca tctgaaattg aatcattcta aagagaagaa aataagaacc 4740 aggtaaaaac tgactagatg ctgccaaatc aaacccaagg tctcctaacc gaaccttaat 4800 gggcctgctt ctctcccagg gcaagtagtt aagagtctca ttccaggaac cctttgtagt 4860 tagttggctg gcatgtttac ttgctgctgt agccagccaa gatgagtgca cctaggccct 4920 caaaaaggat ttttttttac ctcaatctag cctgaccctc atatctgtgg ttgcctctga 4980 gacgaacatc catgctagta taaaaatagt taggaatgcc cttggcagaa ctgaagctct 5040 tattaaatgg tggacagagc tcttgccagc tttgatccag cccctcagtc tcggagttat 5100 ggggcagggt tggggggcag tgctacactg taaagaattt ccaggctggg cgcggtggct 5160 catgcctgta atcccagcac tttgggaggc cgaggagggc ggatcacaag gtcaagagat 5220 tgagatcatc ctggccaaca tggtgaaact ccgtctctac taaaaataca aaaattagct 5280 gggcatggtg gcacgtgcct gtagtcccag ctacttggga ggctgaggca ggagaatcgc 5340 tggaacccag gaggcagaag gggcagtgag ccgagatcgt gccactgcac tccagcctgg 5400 cgacagagcg agactctgtc ttaaaaaaaa aacaaaaaca aaaacaaaaa aaaaaaaaga 5460 atttccagat gtctcttaaa atgtcatgaa taaaattgga aactgtagaa gtgttaatgt 5520 gtcctatgga ctcaatagca gagtttattt ttgtttttaa tggcaaggct tctagagtca 5580 atgattgtat gagtttgcta ctctggctgt gcttacagct tcatccaagt acaagggaag 5640 aaccaatgag gtggattggc tgacagtggg gcatgcccca gcataagccc tagagaggtc 5700 gacagtagtg cagaatgaaa acttggtcgg ggatggcggg tggtgggtgg gaggagggtg 5760 ggctggccgg gagggagaaa tggagtttgt tctacttact gataaagctt tgtgaactaa 5820 ttttatacaa ttcataaaat ttggtgcctt gtactcaatt acggtttgca tggaattgaa 5880 aagattaagg gaaaggattt cttcttgtca tttcaagtgg caaatgggtc tgtgtagtgt 5940 ttaaaggaca tctggtatca cagactttta aaaagcattt catgtctctt gtgtgtgaat 6000 gcaaattatt cttaccagca agttctgaaa ttatgttaca atgtccaaat tctttgtatt 6060 tgtgactgtt catcaaatgg ctgcttttca aggcagactg tgtactccat tgtttttcac 6120 ttcccttgtc atgttccata cattgacttg ggccttttta aatcaggctg gggggctacc 6180 ccttcttaaa tgaatactat atccctatct gaggttacct cttgtgagtg gaagggttgc 6240 tttcccaggt ggttgggaga gcacaaatgt tgagaaaagg ccttttagtc tttatcaaaa 6300 caatgactaa tgctgggcaa catatagcag aagtcaaaca aaaatgattc ctcctgaaat 6360 ggtaccttca ttggttctga tgttcttttg caaaaaatgt ccggaagcag tatagaaaac 6420 cccctgtatg tgaaccttag attgtaaggc aagccagtgc actgtgtaat atgcaaaggg 6480 agtggtgcaa aagagaaggc acccgtccca gctctccatg ccctttccct ggcaggcgct 6540 gactgagatg gcgctgagtg aatgtggaag gtgctaagac ccagtctctt cactgcaggg 6600 ctgtttacag ccagtttctt ttactctgac cttaaggttt ccactgagct catgaaactt 6660 caacatatgg tgtttaaaag tttcccactc aaatgcacac ttgaaaattg ggtccctcag 6720 cctcattact gaactaaata agtgaagaag ttgtatcttt ggggtaaaac tttccagggc 6780 cacttaattt tttaaccagt taagtacgca tcttcctggt ggttcagact ttttaagtca 6840 gtgctaaagc tgaaaagcgc cttctaaata tgactgtgag tttagtttgt atcttacatt 6900 tagtaactgt tctgttgggc caggactatg actttaatta ctatgtactt attcatccca 6960 cactctgcac taaatacacc atagctacta tagcattgtg gcaggcactc atttttgtgg 7020 actcattcca tacttctgct ggcatatttc gtttaaaatc tagctaaaga ttttcatagt 7080 actgtgttaa ctagccaggg ctgaaaagtg tgacaggata tcaaattgac tttccttggg 7140 gaaaatgttg ctttggcaat agtgaagtcc aactccttca tttacagaat tagtgattac 7200 tcatcaattt ttgaaagttg tagattaagt agttttattt actaaattct taaccacttt 7260 gaagatttaa gccagttgaa atgaaacatt ggctggatgc agttggctag cacctgtaat 7320 cccaacactt tgggaggcca aggtgggagg attgcttgag gccaggagct caagaccagc 7380 ctgggaaaca tagatttgaa atcttttcaa aaatgtataa aaatttaata aataaataaa 7440 tgaatcctta ctcaaccata attatggctt taaaacccaa ccaacagtaa acacttactt 7500 gaagccaaaa cagtaaattt tgaatgaaat ttgaattatg cttttggaag ctgccatgtc 7560 ttaattcttg ctggattttg attttcaatt ttggaagttt ttatcaaaac tgtccagtca 7620 tgagtatatc aggtagcagc ctttaaggat ctaagtttca ttcctataaa gaacaggagg 7680 tgctggtatt tcttgtctta gcaagtgatc tgatgcaggt ctaaatgggg gttagcatct 7740 atactatggc ttaaaagttt cctccttggt caccatttgg gaggtgatgg cttcaaagct 7800 aagctttgct ctcatgtgac ccttctttat cttaataccc aataagatat aaacctctca 7860 gagtcaaccc agtgtagact caaagggtct tagcttcatc tatcagataa gatctatctg 7920 atttatacaa agaatggtgc tgtttttata tacatactaa aaaaatctaa ttctcaaaca 7980 attttcttaa tatttatgcc agcaaggtca ggaaagacta ttttgtcctg tttaacttta 8040 gtagtgaaca aactgctaga gacatctgtc atccttaaca ggccactgtg gtccttatat 8100 actgaccctc ctttccaatt aacgtccttt aatcccatac tagttccata agagacttgg 8160 cacataaaag cagtattttt tttaatgcct agtaaactgg ccagaaaaaa atattgccat 8220 ggtggacctg tttctatcca cctctttcta tggctatata agtctataca tgccagttcc 8280 gtacatactc catttccaaa gagcccccaa accctggaca gtagcactac catgaatttt 8340 agggtcacaa aacatttcct tctgaaggaa gcagctgcct cagcagtgtc actttagtga 8400 ccactgccca ggcttacttc actttgcctc cccctacaca tcactgccct ctttctgcca 8460 ccaaacctca ctattccagc aatgatgcct ccctggtgtc tggaaaaccc atctggtgca 8520 ctaaaatgct atgcaacacc aaatggcttc ttcccctaat attgctcaac aatgtaaatt 8580 cttcaagaat aaacttcatg ctaggttaac atcaatgtat ggttacagct tccttttttt 8640 tttttccctt ttaaaatgaa gcaaaccacc tccattaaaa tccatgtttt ggggcttgtt 8700 tttgtctctc aagtgtgaaa tataaatcat gtttttatga tttttgtaaa tctttttaca 8760 ccttgttttt gtaagcaaaa gtttcttatt aaaagaataa aatgacaaaa aaaaaaaaaa 8820 aa 8822 <210> 4 <211> 3194 <212> DNA <213> Homo sapiens <400> 4 cgcgccctgg tgacagctcg cgcgtgctga tgatggcgat tctttggatg ggaagagacc 60 ttgaaaggac ctctagttta tcctgcaact ccagtcaata tcccacttaa atcattccag 120 gtaatagaga agaggaaacc taaccaatga ctgtggaata atatggaaga agatggaaaa 180 aacttgttta atgggttgag aaagtggcga cttctataag acatggatag atgcaaacat 240 gtagggcggt tacggctcgc ccaggaccac tccatcctga accctcagaa gtggtgctgc 300 ttagagtgtg ccaccaccga gtccgtgtgg gcctgcctca agtgctccca cgtggcctgc 360 ggccgctata ttgaggacca cgccctgaaa cactttgagg agacgggaca cccgctagcc 420 atggaagtcc gggatctcta cgtgttctgt tacctgtgca aggactacgt gctcaatgat 480 aacccagagg gggacctgaa gctgctaaga agctccctcc tggcggtccg gggccagaaa 540 caggacacgc cggtgagacg tgggcggacg ctgcggtcca tggcttcggg tgaggacgtg 600 gtcctgccgc agcgcgctcc tcagggacag ccgcagatgc tcacggctct gtggtaccgg 660 cgtcagcgcc tgctggccag gacgctgcgg ctgtggttcg agaagagctc ccggggccag 720 gcgaagctgg agcagcggcg gcaggaggag gccctggagc gcaagaagga ggaggcgcgg 780 aggcggcggc gcgaggtgaa acggcggctg ctggaggagc tggccagcac ccctccgcgc 840 aagagtgcac ggctgctcct gcacacgccc cgcgacgcgg gcccggctgc ctcgcgcccc 900 gccgccctcc ctacctcacg cagagtgccc gccgccacac tcaagctgcg tcgccagccg 960 gccatggccc caggcgtcac gggcctgcgc aacctgggca acacctgcta catgaactcc 1020 atcctccagg tgctcagcca cctccagaag ttccgagaat gtttcctcaa ccttgaccct 1080 tccaaaacgg aacatctgtt tcccaaagcc accaacggga agactcagct ttctggcaag 1140 ccaaccaaca gctcggccac ggagctgtcc ttgagaaatg acagggccga ggcatgcgag 1200 cgggagggct tctgctggaa cggcagggcc tccattagtc ggagtctgga gctcatccag 1260 aacaaggagc cgagttcaaa gcacatttcc ctctgccgtg aactgcacac cctcttccga 1320 gtcatgtggt ccgggaagtg ggccctagtg tcgcccttcg ccatgctcca ctcagtgtgg 1380 agcctgatcc ctgccttccg cggctacgac caacaggacg cgcaggaatt tctctgcgag 1440 ctgctgcaca aggtgcagca ggaactcgag tctgagggca ccacacgccg gatcctcatc 1500 cccttctccc agaggaagct caccaaacag gtcttaaagg tggtgaatac catatttcat 1560 gggcagctgc tcagtcaggt cacatgtata tcatgcaatt acaaatccaa taccattgag 1620 cccttttggg acctatccct ggaattccct gaacgctatc actgcataga aaaggggttt 1680 gtccctttga atcaaacaga gtgcttgctc actgagatgc tggccaaatt cacagagaca 1740 gaggccctgg aagggagaat ctacgcttgt gaccagtgta acagcaaacg acgaaaatcc 1800 aatcccaaac cccttgttct gagtgaagct agaaagcagt taatgatcta cagactacct 1860 caggttctcc ggctgcacct taaaagattc aggtggtctg gccgtaatca tcgagagaag 1920 attggggtcc atgtcgtctt tgaccaggta ttaaccatgg aaccttactg ctgcagggac 1980 atgctctcct ctcttgacaa agagaccttt gcctatgatc tctccgcagt ggtcatgcat 2040 cacgggaaag ggtttggctc aggacactac acagcctatt gctacaacac agagggaggt 2100 gcgtgcgctt tactctgtgg ggtgggggac acggaaaggg gttgatttgt ccacatttta 2160 ttgttttcct tttatttcca tcccatggat tacctagagg gaaattacat acatcaaaaa 2220 tccagtggaa agaattgtga aaattgggtt gggcgccgtg gctcacacct gtaatcctag 2280 cgctttggga ggccgcggtg ggtggatcac ctgaggtcag gagttcgaga ccagcctggc 2340 caacatggtt aaaccccgtc tctactaaaa tagtacaaaa attagccggg cgtggtggtg 2400 ggcactggta atcccaggta ctcaagaggc tgaggcagga gaatcgcttg aacctgggag 2460 gcagaggttg cagtgagcca agattatgcc actgcacttc agcctgggtg acagagcaag 2520 actcttatct caaaaaaaat aaaaagaatt atgaaagttg gattcaaatt ttgggtatat 2580 tactaagtaa caacatatgg ttgttctaag aattaaaagg aactgacatg taaaagtgct 2640 tagaacagga cctaggccct gtgcaatggc tcacacctgt aattccaaca ctttgggagg 2700 cagaggcggg aggattgctt gaggccgagg gttcaagact agcctgggca acatagcgag 2760 actgcatctc tacaaaaaat ttaaaaatta tccaggtgta atggtgtgca cctgtgatcc 2820 tagctacttg ggaggctgag gtgggagaat tgctcaagcc caggagtttg aagttgcaat 2880 gagctatgat ttcaccactg cattctagcc tgggcaacag agtgagatcc tgactcaaat 2940 aaataaataa gaagagcagt tgcctaccac atagtactta ttcagtcagt gttagaggat 3000 attgtagtat tatcaaaagt attggttatt gataatatta ctgttttgtg gtaatgctac 3060 ttctgcacac cccagtttcc ttcttggtta tgcagaaaca gtagaacctg tgtaacctca 3120 aggagatatt gtatgaatct cactagccgc agcttataat aaagccgaga acatttaaaa 3180 aaaaaaaaaa aaaa 3194 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 aggactacgt gctcaatgat aacc 24 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gcaggagcag ccgtgcactc t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ccctgaacgc tatcactgca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 ttggccagca tctcagtgag 20

Claims (11)

In order to provide information necessary for the diagnosis of early menopause caused by formaldehyde, the assay method comprising the step of measuring the expression level of the gene encoding the protein of SEQ ID NO: 1 or 2 in the sample of the subject. delete The analysis method according to claim 1, wherein the sample of the subject is blood or urine. The analysis method according to claim 1 or 3, wherein the measurement of the expression level of the gene measures the amount of mRNA or protein. 5. The assay of claim 4 wherein the determination of the amount of protein is performed by western blotting. The method of claim 4, wherein the mRNA amount is measured by performing RT-PCR or real-time PCR. A kit for diagnosing early menopause induced by formaldehyde comprising a molecule capable of measuring the expression level of a gene encoding a protein selected from the group consisting of the protein of SEQ ID NO: 1 or 2,
An antibody, substrate, ligand, or cofactor to which said molecule specifically binds said protein; Or a kit for diagnosing early menopause induced by formaldehyde, which is a primer having a complementary sequence specific for the gene encoding the protein. delete 8. The kit for diagnosing premenopause induced by formaldehyde according to claim 7, wherein the molecule is labeled with a detectable label. The kit for diagnosing early menopause induced by formaldehyde according to claim 7, wherein the primer is in the form of a microarray immobilized on a substrate. The kit for diagnosing premenopause induced by formaldehyde according to claim 7, wherein the primer has a nucleotide sequence selected from the nucleotide sequences of SEQ ID NOs: 5 to 8. 8.

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