JP2016077234A - Diagnostic kit, diagnostic marker, and detection method for early diagnosis of endometriosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce the burden of the conventional definitive diagnosis of endometriosis on patients.SOLUTION: The invention provides a diagnostic kit for diagnosing endometriosis, the kit having a methylation detection means to quantitatively detect methylation amount in at least one endometriosis associated DNA methylation site in DNA derived from the body fluid specimen of a mammal, a subject.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a diagnostic kit, a diagnostic marker, and a detection method for early diagnosis of endometriosis.

  Changes in the female uterus occur due to changes in the secretion of two female hormones (estrogens and progesterone), each cycle of about 4 weeks. The uterus is a pouch-shaped tissue, the inside of which is covered with the endometrium. When estrogen and progesterone act on the endometrium, their thickness increases, allowing implantation (pregnancy) of fertilized eggs. If pregnancy does not occur, unnecessary endometrium is separated from the blood (menstruation).

  The endometrium usually resides inside the uterus, but endometrial tissue or endometrial-like tissue may occur at sites other than the uterus. Such a condition is called endometriosis. Since the endometrium changes with female hormones, the lesion site of endometriosis also changes. The change of the lesion site caused by female holmo causes symptoms such as bleeding, fever, pain, infertility and adhesion between tissues.

  The mechanism of endometriosis is not clear. In addition, since no radical treatment for endometriosis has been established, treatment for endometriosis depends on coping therapy. Since endometriosis is confirmed by definitive diagnoses such as laparoscopic surgery and histological diagnosis, the diagnosis of endometriosis is a heavy burden on the patient. Patent Document 1 includes a step of determining the expression level of an ovarian endometriosis-related gene in a tissue sample containing an epithelial cell derived from an endometrial cyst collected from a subject. A method for detecting tissue is disclosed.

JP 2005-537027 A

  However, the prior art described in the above literature has room for improvement in the following points. First, since it is necessary to directly collect epithelial cells derived from endometrial cysts from the lesion site of the subject, the technique described in the above literature places a heavy burden on the patient as in the case of conventional definitive diagnosis.

  As described above, the present invention has been made in view of the fact that the conventional diagnosis of endometriosis has a large burden on the patient, and is a diagnostic kit, a diagnostic marker, and a highly effective diagnosis of endometriosis. An object is to provide a detection method.

  The present inventor has searched for a diagnostic marker with a small burden on the patient because the burden on the patient is too large in the conventional diagnosis method. The present inventors focused on the relationship between peripheral blood DNA methylation and endometriosis that has not been clarified so far, and assumed the existence of a peripheral blood DNA methylation region specific to endometriosis. Thus, we tried to select and extract methylated DNA regions as diagnostic molecular marker candidates. A comprehensive DNA methylation analysis was attempted using the peripheral blood DNA of endometriosis patients and the healthy blood. 170 regions of specific methylated DNA were extracted by narrowing down by the DNA methylation variation ratio. From these, we succeeded in identifying 23 regions located on 14 genes that could serve as diagnostic molecular markers. Until now, the existence of diagnostic markers based on DNA derived from endometriosis tissue has been clarified, but the present inventor has revealed for the first time that peripheral blood DNA can be a diagnostic marker for endometriosis. . Based on the above findings, a diagnostic kit, a diagnostic marker and a detection method for endometriosis were developed.

  According to the present invention, there is provided a diagnostic kit for diagnosing endometriosis, the amount of methylation in at least one endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of a subject mammal A diagnostic kit having a methylation detection means for quantitatively detecting is provided. It is demonstrated in the Example mentioned later that the amount of methylation in the endometriosis-related DNA methylation site in the body fluid changes in patients suffering from endometriosis. Therefore, by using this diagnostic kit, early diagnosis of endometriosis and differential diagnosis from other diseases can be performed.

  According to the present invention, there is provided a diagnostic marker for diagnosing endometriosis, comprising a methylation amount in at least one endometriosis-related DNA methylation site in DNA in a body fluid sample of a subject mammal. Provided. This diagnostic marker can serve as a marker that enables early diagnosis of endometriosis and differential diagnosis from other diseases.

  According to the present invention, there is also provided a method for detecting an endometriosis disease state index, wherein methylation is performed in at least one endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of a subject mammal. A detection method comprising the step of quantitatively detecting the amount is provided. According to this detection method, it is possible to detect a disease state index of endometriosis that enables early diagnosis of endometriosis and differential diagnosis from other diseases.

FIG. 1A represents the analyzed 485512 CpG sequences as a pie chart. Of this pie chart, 666 locations are CpG sequences of endometriosis patient DNA, which differs in DNA methylation rate by a factor of 4 or more compared to the DNA of healthy volunteers (control). FIG. 1B represents the 566 CpG sequences of FIG. 1A as a pie chart. In this pie chart, 299 places are Hyper-methylated CpG sequences and 267 places are Hyper-methylated CpG sequences. FIG. 2 represents a CpG sequence (170 positions) of endometriosis patient DNA, which differs in DNA methylation rate by 6 times or more compared with control DNA, as a pie chart. In this pie chart, 87 points are Hyper-methylated CpG sequences and 83 points are Hyper-methylated CpG sequences. FIG. 3A shows the result of cluster analysis for 87 Hyper-methylated CpG sequences. FIG. 3B shows the result of cluster analysis for 83 Hypo-methylated CpG sequences.

Definition Endometriosis Endometriosis is a disease in which endometrial tissue or endometrial-like tissue develops at a site other than the uterus. Endometriosis lesions include, for example, peritoneal lesions, ovarian chocolate cysts And adhesion lesions. Endometriosis is determined by clinical diagnosis (eg, interview, external examination, internal examination, rectal examination, ultrasound tomography and nuclear magnetic resonance imaging (MRI)) and definitive diagnosis (eg, laparoscopic surgery and histology). Have been diagnosed. The term “endometriotic tissue” refers to endometrial tissue formed at sites other than the uterus or tissue similar to the endometrium. The term “related to endometriosis” is a term that means “related to at least endometriosis” and does not exclude other diseases.

Methylation The term “methylation” refers to the modification of methyl groups to various substrates. Methylation includes DNA methylation, RNA methylation, and protein methylation, but unless otherwise indicated, in this specification, it refers to DNA methylation. DNA methylation in animals occurs by the conversion of cytosine-phosphate-guanine (CpG) sequence cytosine to 5-methylcytosine. In plant DNA, cytosine in the CpNpG sequence is methylated. There are about 500,000 CpG sequences on the human genome, most of which are thought to be methylated. The CpG sequence is frequently present near the promoter of the gene, and the region where the CpG sequence is frequently present is called a CpG island. CpG Island ”(1) has a CpG dinucleotide frequency corresponding to the“ measured / expected value ratio ”exceeding 0.6, and (2) the criterion that“ GC content ”exceeds 0.5. Represents a contiguous region of genomic DNA that fills. The length of the CpG island is typically about 0.2 kb to about 1 kb or about 2 kb. It is thought that transcription of a gene is suppressed when a CpG island existing in or near the promoter region is methylated.

Methylation site The term “methylation site” as used herein refers to a location on the genome that is involved in methylation, and includes sites that are methylated and unmethylated under certain conditions. In the present specification, the term “methylation site” and the term “methylation region” can be substituted for each other. The methylation site can also be expressed as a cysteine in the CpG sequence. In the present specification, a site that is originally methylated but not methylated under a specific condition is referred to as a Hyper-methylation site, and a site that is not originally methylated but is methylated under a specific condition is referred to as a Hyper-methylation site. Called. As used herein, specific conditions include endometriosis.

Mammal The term “mammal” as used herein refers to any mammal, such as pupae, domestic animals, pet animals, zoo animals, or sports animals such as dogs, cats, cows, Includes buyu, horses, sheep, rabbits, etc. The mammal is preferably baboon.

Quantitative detection The term “quantitative detection” is used herein to mean detection based on numerical data such as concentration, intensity, mobility, mobility, etc. This may mean detection based on the presence or absence of numerical data by adjustment, threshold setting, or the like.

Overview Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description of omission is abbreviate | omitted.

<Embodiment 1: Diagnostic kit>
This embodiment is a diagnostic kit for diagnosing endometriosis, wherein the amount of methylation in at least one endometriosis-related DNA methylation site in DNA derived from a body fluid sample of a subject mammal is determined. This is a diagnostic kit having a methylation detection means for quantitative detection. By using this diagnostic kit, early diagnosis of endometriosis and differential diagnosis from other diseases can be performed.

  The “endometriosis-related DNA methylation site” in the present embodiment is a site (position) on DNA where the methylation state changes in a patient suffering from endometriosis. Such a site may exist on a specific gene or may exist on a promoter of a specific gene. Such sites may exist in the intergenic region. As demonstrated in the examples described later, in a patient suffering from endometriosis, the methylation amount of the endometriosis-related DNA methylation site in the body fluid is changed. The possibility of endometriosis is judged to be high depending on the presence or absence of DNA methylation sites associated with endometriosis. If the DNA methylation site is a Hyper-methylation site, the patient with endometriosis is not methylated. If it is a Hyper-methylation site, the patient with endometriosis is methylated. Judgment is possible from the fact that However, the present invention is not limited to this, and other symptoms and conditions related to endometriosis are also measured by daily methods well known to physicians in this technical field, and using the diagnostic kit according to the present embodiment It is easily possible to obtain pathological indicators.

  The “methylation detection means” in the present embodiment may be a plurality of detection means. The plurality of detection means may be, for example, different detection means for multifaceted analysis, for example, the same or similar detection means for obtaining statistically reliable data, or a combination thereof.

  Methylation detection includes methylation specific PCR (MSP), bisulfite sequencing, digestion with methylation sensitive restriction enzymes combined with PCR, mixed bisulfite cleavage analysis (COBRA) and methylation sensitive single Qualitative and / or quantitative PCR based techniques such as nucleotide primer extension (Ms-SNuPE) may be used. An apparatus to which such a technique is applied is an example of a methylation detection unit.

  “Body fluid” in the present embodiment includes blood, urine, saliva and exudate, but is not limited thereto as long as it contains detectable DNA. In this embodiment, the body fluid is preferably blood.

  In the diagnostic kit of the present embodiment, the methylation amount of the endometriosis-related DNA methylation site may be indicated as a disease state index of the endometriosis. The state of methylation at a specific methylation site can be expressed as a quantitative ratio of methylated sites to unmethylated sites in a DNA sample containing a plurality of such specific methylated sites. Therefore, the state of methylation can be determined by a change in the amount of methylation. As demonstrated in the examples described later, in a patient suffering from endometriosis, the methylation amount of the endometriosis-related DNA methylation site in the body fluid is changed. Therefore, for example, when the amount of methylation at the methylation site is quantitatively detected and a change is confirmed, it is determined that the possibility of endometriosis is high.

  The “disease state index of endometriosis” in the present embodiment refers to an index related to the endometriosis state including, for example, endometriosis or endometriosis-related diseases. Here, endometriosis pathological indicators include, for example, prediction of endometriosis risk, early diagnosis / prediction of endometriosis, endometriosis progression, pathologic condition, progress Indices such as prediction of outcome, observation / evaluation of treatment results, and prediction of prognosis. Further, a plurality of endometriosis condition indicators can be combined. Thereby, a more reliable diagnosis result can be obtained. The symptom and the parameter representing the symptom can be easily measured by a routine method well known to doctors in this technical field.

  “Significant” in this embodiment means that the p-value (U test, two-sided test) is preferably <0.05, more preferably <0.01, and most preferably <0.001.

  In the diagnostic kit of the present embodiment, the disease state index may be a methylation amount of the endometriosis-related DNA methylation site with respect to a preset methylation amount threshold. By contrasting with the threshold, it becomes possible to diagnose endometriosis quickly and accurately.

  For example, if the amount of methylation at the DNA methylation site as a hypo-methylation site is significantly reduced below a threshold, the subject is diagnosed as having a high possibility of endometriosis. When the methylation amount of the DNA methylation site as a hyper-methylation site is significantly increased from the threshold value, the subject is diagnosed as having a high possibility of endometriosis. However, the present invention is not limited to this, and other symptoms and conditions related to endometriosis are also measured by daily methods well known to physicians in this technical field, and using the diagnostic kit according to the present embodiment It is easily possible to obtain pathological indicators.

  The “threshold value” in the present embodiment may be a predicted value, and is preferably a value measured in advance. When using a value measured in advance, a healthy mammal of the same species as the subject is preferred.

  In the diagnostic kit of the present embodiment, the threshold value is set based on the methylation amount of the endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of the same species and healthy mammal as the subject. Also good. By adopting the above methylation amount derived from a healthy mammal of the same species as the subject as a control, it becomes possible to reliably diagnose endometriosis.

  For example, if the methylation level of the DNA methylation site as a hypo-methylation site is significantly less than the methylation level from the healthy mammal, the subject may have endometriosis. Is diagnosed as high. In addition, if the methylation level of the DNA methylation site as a hyper-methylation site is significantly higher than the methylation level derived from the healthy mammal, the subject may have endometriosis. Is diagnosed as high. The threshold value may be the methylation amount obtained from the subject previously. For example, by using the amount of methylation when the subject was healthy as a threshold, it is possible to detect changes over time in the amount of methylation, and more accurately diagnose endometriosis. It becomes possible. However, the present invention is not limited to this, and other symptoms and conditions related to endometriosis are also measured by daily methods well known to physicians in this technical field, and using the diagnostic kit according to the present embodiment It is easily possible to obtain pathological indicators.

  In the diagnostic kit of the present embodiment, the endometriosis-related DNA methylation site may include a Hyper-methylation site and / or a Hyper-methylation site. And, the hyper-methylation site is (1) the position of the first chromosome 231820076, (2) the position of the fifth chromosome 65888284, (3) the position of the sixth chromosome 32552152, (4) the position of the sixth chromosome 32632694, It may comprise a methylation site located at (5) position 1434741 of chromosome 14 and (6) position 3771880 of chromosome 19. The Hypo-methylation site is (1) the position of the second chromosome 43451868, (2) the position of the second chromosome 108157524, (3) the position of the second chromosome 124971544, (4) the position of the fourth chromosome 77997420, (5) position of chromosome 6 32589203, (6) position of chromosome 632522400, (7) position of chromosome 632522872, (8) position of chromosome 632552515, (9) position of chromosome 6 32554385, (10) Chromosome position 32557970, (11) Chromosome position 32558175, (12) Chromosome position 32607174, (13) Chromosome position 32610971, (14) Chromosome position 1225813494 , (15) the position of chromosome 15 at 66947066, (16) the position of chromosome 15 at 90115219, and (17) the position of chromosome 20 at position 47026467. By specifying the methylation site to be detected, endometriosis can be diagnosed quickly and accurately.

  In the present embodiment, detection of methylation at the methylation site may be performed for one methylation site (position) or may be performed for a plurality of methylation sites. The detection of methylation at the Hyper-methylation site may be performed on one, two, three, four, five, or six sites. The detection of methylation at the above-mentioned Hypo-methylation site is performed at 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites. You may perform with respect to a site of 14 places, 15 places, 16 places, or 17 places. When methylation detection is performed on at least one site of the Hyper-methylation site, detection of methylation at the Hyper-methylation site may or may not be performed. Conversely, when detection of methylation is performed on at least one site of the Hyper-methylation site, detection of methylation at the Hyper-methylation site may or may not be performed. . For example, if endometriosis can be diagnosed only by detecting methylation at the hyper-methylation site, detection of methylation at the hyper-methylation site may not be performed, and vice versa. It is. In addition, after detecting methylation at the Hyper-methylation site, detection of methylation at the Hyper-methylation site may be performed, and vice versa. Furthermore, the detection of methylation at the Hyper-methylation site and the Hyper-methylation site may be performed substantially simultaneously using high-throughput processing.

  In the present embodiment, methylation sites other than the methylation sites may be detected. For example, detection of methylation at the 6 hyper-methylation sites (and / or 17 hyper-methylation sites) described above and one or more methylation sites different from the above methylation sites May be performed. Moreover, you may combine the amount of methylation in the said methylation site, and the quantity of a known marker. An example of the known marker is tumor marker CA-125. The amount of the known marker may be a concentration or an active amount as long as endometriosis can be diagnosed by combining with the methylation amount. When the methylation amount and the known marker are combined, the diagnostic kit may further have a known marker detection means. In addition, a plurality of known markers can be adopted as the known marker, and a plurality of known marker detection means matched to each known marker can be prepared. Examples of the detection means include an enzyme immunoassay method using a specific antibody specific to a marker, a Western blot method, an antibody array, and the like, and a plurality of combinations can be used.

  In the diagnostic kit of this embodiment, the at least one endometriosis-related DNA methylation site may be all the methylation sites included in the Hyper-methylation site and the Hyper-methylation site. . By detecting methylation at the 23 types of methylation sites, it is possible to diagnose endometriosis with higher accuracy even in the case of early endometriosis.

  For example, the methylation level of all of the six types of Hyper-methylation sites in the subject is significantly greater than the methylation level of all of the six types of Hyper-methylation sites in a healthy mammal, and When the methylation level of all of the 17 types of Hypo-methylation sites in the subject is significantly less than the methylation level of all of the 17 types of Hypo-methylation sites in a healthy mammal, The subject is diagnosed with a very high probability of endometriosis.

  In the diagnostic kit of the present embodiment, the methylation amount may be a methylation rate. Since a methylation rate can be easily calculated by using a general methylation detection method, endometriosis can be easily diagnosed.

  In this embodiment, for example, when the methylation rate of a hyper-methylation site in a subject is preferably 6 times or more larger than the methylation rate of the same hyper-methylation site in a healthy mammal, Hyper-methylation sites in the body may be determined to be methylated. Also, for example, if the methylation rate of a hypo-methylation site in a healthy mammal is preferably more than 6 times greater than the methylation rate of the same hypo-methylation site in the subject, the hypo in the subject -Methylation sites may be determined not to be methylated.

  In the diagnostic kit of the present embodiment, the body fluid sample may be blood. Compared with a conventional method for diagnosing endometriosis, in which endometriosis tissue is directly collected and examined, it is possible to diagnose endometriosis with a significantly reduced burden on the patient.

  In the present embodiment, the peripheral blood DNA obtained from the blood of the subject may be DNA obtained from whole blood or DNA obtained from isolated leukocytes. Regarding blood, it is also possible to use serum that has been subjected to appropriate centrifugation and reduced to a negligible amount of lymphocytes. In the present embodiment, it is possible to react a detection reagent with the serum.

  Here, one or a plurality of arbitrary processing steps may be added to the obtained sample such as blood or serum. Such treatment steps include, for example, removal of impurities by various columns and immunoprecipitation methods, purification of the target product, protein fragmentation by lipase, etc., separation of contaminants by enzyme treatment or chemical treatment, Examples include, but are not limited to, enzyme treatment and various chemical modifications.

<Embodiment 2: Diagnostic marker>
In addition, other embodiments of the present invention diagnose endometriosis comprising the amount of methylation in at least one endometriosis-related DNA methylation site in DNA in a body fluid sample of a subject mammal It is a diagnostic marker for. This diagnostic marker can serve as a marker that enables early diagnosis of endometriosis and differential diagnosis from other diseases.

  As demonstrated in the examples described later, in a patient suffering from endometriosis, the methylation amount of the endometriosis-related DNA methylation site in the body fluid is changed. Therefore, for example, by using the amount of methylation at the endometriosis-related DNA methylation site as a diagnostic marker, the role as a marker indicating that the possibility of endometriosis is high from the change in the amount of methylation The endometriosis can be diagnosed based on the change in the amount of methylation. However, the present invention is not limited to this, and other symptoms / conditions related to endometriosis are also measured by daily methods well known to doctors in this technical field, and they are measured using the diagnostic marker according to the present embodiment. Diagnosis is easily possible.

  The diagnostic marker of the present embodiment is the subject with respect to the methylation amount of the endometriosis-related DNA methylation site in DNA in a body fluid sample obtained from the same species and healthy mammal as the subject. A methylation amount of at least one endometriosis-related DNA methylation site in DNA in a mammalian body fluid sample may be included. By adopting the methylation amount derived from a healthy mammal of the same species as the mammal as a control, it becomes a diagnostic marker that can be used for reliable diagnosis of endometriosis.

  In the diagnostic marker of this embodiment, the endometriosis-related DNA methylation site may include a Hyper-methylation site and / or a Hyper-methylation site. And, the hyper-methylation site is (1) the position of the first chromosome 231820076, (2) the position of the fifth chromosome 65888284, (3) the position of the sixth chromosome 32552152, (4) the position of the sixth chromosome 32632694, It may comprise a methylation site located at (5) position 1434741 of chromosome 14 and (6) position 3771880 of chromosome 19. The Hypo-methylation site is (1) the position of the second chromosome 43451868, (2) the position of the second chromosome 108157524, (3) the position of the second chromosome 124971544, (4) the position of the fourth chromosome 77997420, (5) position of chromosome 6 32589203, (6) position of chromosome 632522400, (7) position of chromosome 632522872, (8) position of chromosome 632552515, (9) position of chromosome 6 32554385, (10) Chromosome position 32557970, (11) Chromosome position 32558175, (12) Chromosome position 32607174, (13) Chromosome position 32610971, (14) Chromosome position 1225813494 , (15) the position of chromosome 15 at 66947066, (16) the position of chromosome 15 at 90115219, and (17) the position of chromosome 20 at position 47026467. By using a specific methylation site, it becomes a diagnostic marker that can be used for accurate diagnosis of endometriosis.

  In the diagnostic marker of the present embodiment, the at least one endometriosis-related DNA methylation site may be all the methylation sites included in the Hyper-methylation site and the Hyper-methylation site. . By using all of the 23 types of methylation sites, it becomes a diagnostic marker that can be used for the diagnosis of early endometriosis.

<Embodiment 3: Detection method>
In another embodiment of the present invention, there is provided a method for detecting an endometriosis condition indicator, wherein at least one endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of a subject is a subject. It is a detection method including the process of detecting the amount of methylation in Quantitatively. According to this detection method, it is possible to detect a disease state index of endometriosis that enables early diagnosis of endometriosis and differential diagnosis from other diseases.

  As demonstrated in the examples described later, in a patient suffering from endometriosis, the methylation amount of the endometriosis-related DNA methylation site in the body fluid is changed. Therefore, for example, by quantitatively detecting the amount of methylation, a change in the amount can be detected. Based on this result, a material judged to have a high possibility of endometriosis is obtained. However, the present invention is not limited to this, and other symptoms / conditions related to endometriosis are also measured by daily methods well known to doctors in this technical field, and the detection method according to the present embodiment is used to measure them. Judgment is easily possible.

  Further, the detection method of the present embodiment is a step of determining whether or not the methylation amount of the endometriosis-related DNA methylation site is significantly changed with respect to a preset methylation amount threshold value. May further be included. By contrasting with the threshold, it becomes possible to diagnose endometriosis quickly and accurately.

  In the detection method of the present embodiment, the threshold is set based on the methylation amount of the endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of the same species and healthy mammal as the subject. May be. By adopting the above methylation amount derived from a healthy mammal of the same species as the subject as a control, it becomes possible to reliably diagnose endometriosis.

  In the detection method of the present embodiment, the endometriosis-related DNA methylation site may include a Hyper-methylation site and / or a Hyper-methylation site. And, the hyper-methylation site is (1) the position of the first chromosome 231820076, (2) the position of the fifth chromosome 65888284, (3) the position of the sixth chromosome 32552152, (4) the position of the sixth chromosome 32632694, It may comprise a methylation site located at (5) position 1434741 of chromosome 14 and (6) position 3771880 of chromosome 19. The Hypo-methylation site is (1) the position of the second chromosome 43451868, (2) the position of the second chromosome 108157524, (3) the position of the second chromosome 124971544, (4) the position of the fourth chromosome 77997420, (5) position of chromosome 6 32589203, (6) position of chromosome 632522400, (7) position of chromosome 632522872, (8) position of chromosome 632552515, (9) position of chromosome 6 32554385, (10) Chromosome position 32557970, (11) Chromosome position 32558175, (12) Chromosome position 32607174, (13) Chromosome position 32610971, (14) Chromosome position 1225813494 , (15) the position of chromosome 15 at 66947066, (16) the position of chromosome 15 at 90115219, and (17) the position of chromosome 20 at position 47026467. By specifying the methylation site to be detected, endometriosis can be diagnosed quickly and accurately.

  In the detection method of the present embodiment, the at least one endometriosis-related DNA methylation site is the Hyper-methylation site and all the methylation sites included in the Hyper-methylation site. Also good. By detecting methylation at the 23 types of methylation sites, it is possible to diagnose endometriosis with higher accuracy even in the case of early endometriosis.

  As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable.

  For example, in the above embodiment, the diagnostic kit, the diagnostic marker, and the detection method are used. However, the endometriosis diagnosis method, the endometriosis tissue detection method, and the method for providing information for assisting the diagnosis of endometriosis It is good. In this way, it is possible to diagnose endometriosis using the body fluid of the subject, detect endometriosis tissue, and provide information that assists in the diagnosis of endometriosis.

  EXAMPLES Hereinafter, although an Example and drawing demonstrate this invention further, this invention is not limited to these. The instrument operation and kit use were in accordance with the manufacturer's manufacturer's protocol.

<Example 1>
Peripheral blood DNA preparation from endometriosis patients Peripheral blood from endometriosis patients (4 patients) and healthy volunteers (4 persons) who received a written consent from a female medical department at Tottori University Hospital 2 ml of was collected. The age of patients and volunteers was 38 ± 3 years. Peripheral blood DNA was prepared from whole blood using DNeasy Blood & Tissue Kit (QIAGEN).

<Example 2>
Methylation450 array analysis After the prepared DNA was treated with bisulfite, the CpG islands contained in the human gene 21231 and the surrounding 4855512 CpG sequences in the Infinium HumanMethylation450 BeadChip (illumina) The conversion rate was measured.

<Example 3>
Analysis of Methylation450 array analysis data by GeneSpringGX
The data of Methylation450 array was analyzed using GeneSpringGX. 556 CpG sequences of endometriosis patient DNA, in which the DNA methylation rate differs by 4 times or more compared with the DNA of healthy volunteers (control), were extracted (FIG. 1A). These included 299 Hyper-methylated CpG sequences and 267 Hyper-methylated CpG sequences (FIG. 1B).

  Furthermore, 170 CpG sequences of endometriosis patient DNA, which differed by 6 times or more in DNA methylation rate compared with control DNA, were extracted. These included 87 Hyper-methylated CpG sequences and 83 Hyper-methylated CpG sequences (FIG. 2).

  Cluster analysis was performed on 170 CpG sequences in endometriosis patient DNA. As a result, 87 Hyper-methylated CpG sequences were classified into 3 groups of clusters (16, 20 and 51 CpG sequences) (FIG. 3A), and 83 Hyper-methylated CpG sequences were grouped into 3 groups. Cluster (20, 21 and 42 CpG sequences) (FIG. 3B).

  Next, in all endometriosis patients, CpG sequences of endometriosis patient DNAs having a DNA methylation rate different by 6 times or more compared with control DNA were extracted. As a result, 23 CpG sequences located on 14 genes were selected as diagnostic marker candidates from the 170 CpG sequences in the endometriosis patient DNA. Six Hyper-methylated CpG sequences located on 5 genes and 17 Hyper-methylated CpG sequences located on 9 genes were finally selected. Table 1 shows a list of positions on the genome of Hyper-methylated CpG sequences, and Table 2 shows a list of positions on the genome of Hyper-methylated CpG sequences.

  From the above results, regarding the methylation rates of all Hypo-methylation sites listed in Table 1, the methylation rates of healthy subjects were at least 6 times higher than the methylation rates of endometriosis patients, respectively. . In addition, regarding the methylation rates of all the Hyper-methylation sites shown in Table 2, each methylation rate of endometriosis patients was at least 6 times higher than that of healthy individuals.

<Discussion>
From the results of this example, the methylation rate of endometriosis-related DNA methylation sites (see Tables 1 and 2) in the peripheral blood DNA of endometriosis patients is different from that of healthy subjects. It became clear. Endometriosis can be diagnosed by quantitatively detecting the amount of methylation at endometriosis-related DNA methylation sites in DNA in a body fluid sample.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (17)

A diagnostic kit for diagnosing endometriosis,
A diagnostic kit comprising a methylation detection means for quantitatively detecting the amount of methylation in at least one endometriosis-related DNA methylation site in DNA derived from a body fluid sample of a mammalian subject.   The diagnostic kit according to claim 1, wherein the amount of methylation at the endometriosis-related DNA methylation site is indicated as a disease state index of the endometriosis.   The diagnostic kit according to claim 2, wherein the pathological condition index is a methylation amount of the endometriosis-related DNA methylation site with respect to a preset methylation amount threshold.   The diagnosis according to claim 3, wherein the threshold value is set based on a methylation amount of the endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of the same species and healthy mammal as the subject. kit. The endometriosis-related DNA methylation site includes a Hyper-methylation site and / or a Hyper-methylation site,
The Hyper-methylation site is
(1) position of the first chromosome 231820076,
(2) Location of chromosome 5 65888284,
(3) Chromosome 6 position 32552152,
(4) Chromosome 6 position 32632694,
(5) Chromosome 14 position 70347441, and
(6) comprising a methylation site located at position 3771880 of chromosome 19,
The Hypo-methylation site is
(1) second chromosome position 43451868,
(2) second chromosome position 108157524,
(3) second chromosome position 124971544,
(4) Chromosome 4 position 77997420,
(5) position of chromosome 6 3289203,
(6) position 6522400 of chromosome 6,
(7) location of chromosome 632522872,
(8) position 6325515 of chromosome 6,
(9) Location of chromosome 6 32554385,
(10) Location of chromosome 6 32557970,
(11) location of chromosome 6 32558175,
(12) Chromosome 6 position 32607174,
(13) Position 6310971 of chromosome 6,
(14) Location of chromosome 1212513494,
(15) Location of chromosome 15 66947066,
(16) Chromosome 15 position 90115219, and
(17) The diagnostic kit according to any one of claims 1 to 4, comprising a methylation site located at position 47026467 of chromosome 22;   The diagnostic kit according to claim 5, wherein the at least one endometriosis-related DNA methylation site is all of the methylation sites contained in the Hyper-methylation site and the Hyper-methylation site.   The diagnostic kit according to claim 1, wherein the methylation amount is a methylation rate.   The diagnostic kit according to claim 1, wherein the body fluid sample is blood.   A diagnostic marker for diagnosing endometriosis, comprising the amount of methylation in at least one endometriosis-related DNA methylation site in DNA in a body fluid sample of a subject mammal.   DNA in the body fluid sample of the subject mammal against the methylation amount of the endometriosis-related DNA methylation site in DNA in the body fluid sample obtained from the same species and healthy mammal as the subject The diagnostic marker according to claim 9, comprising a methylation amount of at least one endometriosis-related DNA methylation site in. The endometriosis-related DNA methylation site includes a Hyper-methylation site and / or a Hyper-methylation site,
The Hyper-methylation site is
(1) position of the first chromosome 231820076,
(2) Location of chromosome 5 65888284,
(3) Chromosome 6 position 32552152,
(4) Chromosome 6 position 32632694,
(5) Chromosome 14 position 70347441, and
(6) comprising a methylation site located at position 3771880 of chromosome 19,
The Hypo-methylation site is
(1) second chromosome position 43451868,
(2) second chromosome position 108157524,
(3) second chromosome position 124971544,
(4) Chromosome 4 position 77997420,
(5) position of chromosome 6 3289203,
(6) position 6522400 of chromosome 6,
(7) location of chromosome 632522872,
(8) position 6325515 of chromosome 6,
(9) Location of chromosome 6 32554385,
(10) Location of chromosome 6 32557970,
(11) location of chromosome 6 32558175,
(12) Chromosome 6 position 32607174,
(13) Position 6310971 of chromosome 6,
(14) Location of chromosome 1212513494,
(15) Location of chromosome 15 66947066,
(16) Chromosome 15 position 90115219, and
(17) The diagnostic marker according to claim 9 or 10, comprising a methylation site located at position 47026467 of the twenty-second chromosome.   The diagnostic marker according to claim 11, wherein the at least one endometriosis-related DNA methylation site is all the methylation sites included in the Hyper-methylation site and the Hyper-methylation site. A method for detecting a disease state index of endometriosis,
A detection method comprising a step of quantitatively detecting the amount of methylation in at least one endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of a subject mammal.   The method according to claim 13, further comprising a step of determining whether or not a methylation amount of the endometriosis-related DNA methylation site is significantly changed with respect to a preset methylation amount threshold. Detection method.   The threshold value is set based on the methylation amount of the endometriosis-related DNA methylation site in DNA derived from a bodily fluid sample of the same species and healthy mammal as the subject. Detection method. The endometriosis-related DNA methylation site includes a Hyper-methylation site and / or a Hyper-methylation site,
The Hyper-methylation site is
(1) position of the first chromosome 231820076,
(2) Location of chromosome 5 65888284,
(3) Chromosome 6 position 32552152,
(4) Chromosome 6 position 32632694,
(5) Chromosome 14 position 70347441, and
(6) comprising a methylation site located at position 3771880 of chromosome 19,
The Hypo-methylation site is
(1) second chromosome position 43451868,
(2) second chromosome position 108157524,
(3) second chromosome position 124971544,
(4) Chromosome 4 position 77997420,
(5) position of chromosome 6 3289203,
(6) position 6522400 of chromosome 6,
(7) location of chromosome 632522872,
(8) position 6325515 of chromosome 6,
(9) Location of chromosome 6 32554385,
(10) Location of chromosome 6 32557970,
(11) location of chromosome 6 32558175,
(12) Chromosome 6 position 32607174,
(13) Position 6310971 of chromosome 6,
(14) Location of chromosome 1212513494,
(15) Location of chromosome 15 66947066,
(16) Chromosome 15 position 90115219, and
(17) The detection method according to any one of claims 13 to 15, comprising a methylation site located at position 47026467 of the twenty-second chromosome.   The detection method according to claim 16, wherein the at least one endometriosis-related DNA methylation site is all the methylation sites contained in the Hyper-methylation site and the Hyper-methylation site.