KR101069139B1 - A kit and a method for detecting biomarrkers for down's syndrome - Google Patents

Abstract

The present invention discloses a detection kit and detection method for Down syndrome biomarkers. The detection kit and detection method of the present invention are characterized by being able to detect an unmethylated PDE9A gene fragment comprising the sequence of SEQ ID NO: 2, a Down syndrome biomarker.

Description

A KIT AND A METHOD FOR DETECTING BIOMARRKERS FOR DOWN'S SYNDROME}

The present invention relates to detection kits and methods of detecting Down syndrome biomarkers.

Down Syndrome is a chromosome disease in which chromosome 21 is present in three more than one normal person. Cytogenetically referred to as chromosome 21.

Down's syndrome shows abnormalities throughout the body, including mental retardation, physical malformations, systemic dysfunction, growth disorders, and shorter lifespan than the general population.

The number of births for Down syndrome is about 1 in every 800 people, and there are no significant differences in race or socioeconomic class, but gender is slightly higher for males (3: 2).

Prenatal screening methods for Down's syndrome include nucleus translucency ultrasound scan of the fetus and serum markers (pregnancy-associated plasma protein A, human chorionic gonadotrophin, alpha fetoprotein, unconjugated estriol, and inhibin-A). The combination test method of the is parallel. In particular, the diagnostic accuracy of Down's syndrome using fetal neck translucent ultrasonography (PAPPA), pregnancy-associated plasma protein A (PAPPA), and serum markers of human chorionic gonadotrophin (HCG) during the first trimester was 22-63 under 5% false-positive rate. Very low in% and reported to be approximately 84% accurate under 5% false positive rate when other methods are evaluated in combination with the method (Obstet Gynecol. 2006; 107: 167-173, N Engl J Med) 2005; 353: 2001-11).

In general, accurate diagnosis of Down's syndrome is performed by chromosome analysis through amniocentesis or chorionic villus sampling.However, such amniocentesis or chorionic biopsy is an invasive method and has a risk of abortion rate of approximately 1%. It is known to have.

Therefore, non-invasive screening and diagnostic techniques with high accuracy for Down syndrome are still required to reduce the need for current invasive diagnostic methods and to replace screening methods.

It is an object of the present invention to provide a detection kit and detection method for Down syndrome biomarkers, which are useful for diagnosing Down syndrome non-invasively using the blood of a pregnant woman.

Other and specific objects of the present invention will be presented below.

Fetal DNA present in pregnant women's blood originates from the placenta, a fetal tissue, and is known to occur during death following the spontaneous apoptosis of the trophoblast that occurs during pregnancy and release into the pregnant woman's blood (Prenat Diagn 2007; 27: 415-8., Am J Pathol 2006; 169: 400-4).

The fact that fetal DNA is present in the blood of pregnant women has been reported for the first time by detecting the specific DNA of the Y chromosome in the blood of pregnant women with male fetus (Lancet 1997; 350: 485-487). It is being researched as a new source of non-invasive prenatal diagnosis of hereditary diseases in children and fetuses.

Fetal DNA in pregnant women's blood is known to increase in pregnancy-related diseases caused by abnormal placental conditions such as preeclampsia and fetal growth development disorders (Am J Pathol 2006; 169: 400-4; Prenat Diagn 2007; 27: 415). -8; Genes Dev 2000; 14: 3191-203; Am J Obstet Gynecol 2009; 200: 98.e1-6; Human Reproduction 2003; 18 (8): 1733-736), even in fetal genetic diseases such as Down's syndrome. It is reported to increase (Am J Obstet Gynecol. 2002 Nov; 187 (5): 1217-21. Clin Chem. 2003; 49: 239-242. Clin Chem 1999; 45: 1747-51).

However, the specific DNA sequence of the Y chromosome used in previous studies related to Down syndrome using fetal DNA in pregnant women's blood has limitations that cannot be applied when the fetus is a girl. Under a very low disadvantage of 21% (Am J Obstet Gynecol. 2002 Nov; 187 (5): 1217-21. Clin Chem. 2003; 49: 239-242. Clin Chem 1999; 45: 1747-51). Therefore, the epigenetic characteristics of genes such as DNA methylation have been attracting attention in order to more accurately measure fetal DNA present in maternal blood regardless of the sex of the fetus.

Down's syndrome is generally a disease that occurs because there are three chromosomes 21.

Recently, DNA sequences showing different methylation patterns in blood cells and placental tissues of chromosome 21 were identified (Clin Chem. 2008; 54: 500-511). Clin Chem. 2008; 54: 500-511 are considered part of this specification.

Among the DNA sequences thus identified, some of the sequences of the PDE9A gene (GenBank Accession No. AB017602.1) (the sequence referred to as 084 CGI in Clin Chem. 2008; 54: 500-511) are This part has been confirmed to be completely methylated in the blood cells of pregnant women, but unmethylated in the trophoblast (Clin Chem. 2008; 54: 500-511). Here, "fully methylated" means that the methylation index is defined as methylation level / (methylation level + unmethylation level) in evaluating the degree of methylation of a gene. Methylated "means that the methylation index has a value of 1 or less (the methylation index is specifically defined in Clin Chem. 2008; 54: 500-511). For this sequence identified (the sequence referred to as 084 CGI in Clin Chem. 2008; 54: 500-511), it exhibits a value of 0.5 or less in placental cells during early pregnancy, which is 50% for placental-derived DNA. It means that the above is unmethylated.

DNA methylation is a histone transform as one of the Ministry of chemical change (epigenetic modification), such as (histone modification), a methyl group by a DNA methylation enzyme (DNA methyl transferase) in CpG islands (CpG islands) with a CpG motif, together multiple (CH ₃ ) Is combined. Since CpG islands are mainly located at the beginning of promoters or exons, methyl group binding to CpG islands is generally known to interfere with mRNA transcription and inhibit gene expression (Semin Cancer Biol 1999; 9 (5): 349-57).

In previous reports, the PDE9A gene was highly expressed in tissues such as the brain, heart, and placenta but not in blood cells (Hum Genet. 1998; 103: 386-392). It seems to be the effect of. Moreover, the positional feature of the PDE9A gene on chromosome 21 is suggested to be related to the overexpression of this gene by the addition of chromosome 21 and the development of Down syndrome (Hum Genet. 1998; 103: 386-). 392).

Based on the results of these previous studies, the present inventors have identified 18 pregnant women who are pregnant with down syndrome fetuses of first-trimester, and pregnant women who are pregnant with normal fetuses, as confirmed in the following examples. Blood was collected from the blood, and plasma was separated. Nucleic acid samples were extracted from the plasma with a high percentage of fetal DNA, followed by methylation specific PCR (Methylation specific PCR). When pregnant blood cells die , their genomic DNA fragments are released into the blood of the pregnant woman) Methylated PDE9A gene fragment (hereinafter referred to as "methylated PDE9A gene fragment") and unmethylated PDE9A gene fragment which appears to be derived from placental cells (hereinafter The concentration in plasma of the "unmethylated PDE9A gene fragment" is determined. Methylated PDE9A gene fragment Total 69bp in 35552nd nucleotide to the 35620th nucleotide (SEQ ID NO: 1) was used as a primer capable of amplifying, unmethylated PDE9A gene fragment Total in 35543rd nucleotide to the 35621st nucleotide 79bp (SEQ ID NO: 2 Primers capable of amplifying ( PDE9A gene fragment) were used. These primers were designed and produced based on the nucleic acid sequence after treatment with bisulfite, which transforms cytosine of unmethylated nucleic acid into uracil.

As a result, the concentration of methylated PDE9A gene fragment was not significantly different between pregnant women pregnant with Down syndrome fetus and its control group, while the concentration of unmethylated PDE9A gene fragment was not found between pregnant women pregnant with Down syndrome fetus and its control group. Significant differences were found.

Also was unmethylated PDE9A a diagnostic accuracy (AUC, Area Under Curve) when writing the ROC curve (receiver operating characteristic curve) based on the concentration of the gene fragment 0.941, [(unmethylated PDE9A concentration of gene fragment) / (methylated PDE9A concentration + unmethylated PDE9A concentration of gene fragment) of the gene fragment ( "non-methylation index (unmethylation index") diagnostic accuracy based on the combined value of the density of the density and methylation PDE9A gene fragment of the four unmethylated PDE9A gene fragment including the (AUC, Area Under Curve) were both 0.970 (where, Im [unmethylated PDE9A density / methylated PDE9A concentration in the blood in the blood] is 0.969).

It is well known in the art that the diagnostic accuracy, that is, the area under the curve of the ROC curve is 0.9-1 is a very good test method, 0.8-0.9 is a good test method, and 0.7-0.8 is a moderate test method.

And when the cut-off value of the unmethylated PDE9A gene fragment was 415 genome equivalent (GE) / mL, the specificity was 0.944, the sensitivity was 0.778, and the positive predicative value. ), 0.737 and negative predicative value were 0.955. When the cut value was 140 in the non-methylation index, the specificity was 0.944, the sensitivity was 0.833, the positive predictive value was 0.750, and the negative predictive value was 0.966. Genome equivalents (GE) are described in Clin Chem. 2001; 47: 1607-1613; Am J Hum Genet. 1998; 62: 768-775; Am J Hum Genet 1999; 64: 218-24.

These experimental results can be said that the unmethylated PDE9A gene fragment is a biomarker that can be useful for the diagnosis of pregnant women with Down syndrome.

As used herein, "non-methylated PDE9A gene fragment" refers to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 as some sequences in the PDE9A gene. Considering that fetal DNA, which is usually present in maternal blood, is reported to be 99% or more in length and 313 bp or less, and 80% or more is reported to be 193 bp or less (Clin Chem. 2004; 50: 88-92). The non-methylated PDE9A gene fragment refers to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 and having a length of 313 bp or less, preferably 193 bp or less. Most preferably refers to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.

The unmethylated PDE9A gene fragment is referred to herein as Down syndrome biomarker for convenience.

The Down syndrome biomarker of the present invention is present in a processed sample of blood of a pregnant woman with Down syndrome fetus, or blood of pregnant women, such as plasma, is present in a higher concentration than the processed sample of blood of a normal fetal pregnant woman, or plasma of pregnant women. Therefore, the value determined for the concentration of the Down syndrome biomarker of the present invention present in the processed sample of pregnant woman blood, such as maternal blood or plasma, is determined independently (i.e. by itself) or in combination with known Down syndrome diagnosis methods. It can be useful for diagnosing pregnant women with Down syndrome. Known Down syndrome diagnosis includes fetal neck umbilical cord ultrasonography as described above, a test method using a serum labeling material such as MSAFP (maternal serum alpha fetoprotein), amniotic fluid, chorionic biopsy, and the like. And other known methods such as a method using an integrated microarray (Korean Patent Registration No. 10-0804417).

In summary, the present invention may be understood as a kit for detecting a Down syndrome biomarker including a probe and / or a primer specifically binding to the Down syndrome biomarker.

The probe or primer included in the kit for detecting the Down syndrome biomarker of the present invention specifically binds to the Down syndrome biomarker of the present invention, hybridizes, or acts as an initiation point of a nucleic acid amplification reaction. It can be used to detect, qualitatively and quantitatively analyze the Down syndrome biomarker of the present invention in processed samples of maternal blood such as plasma.

As used herein, "processed sample of maternal blood" means plasma or serum as a sample derived (obtained) from blood by centrifugation or the like.

Because fetal DNA in maternal blood is known to be present in relatively higher amounts in plasma than in serum (Am J Hum Genet 1998b; 62: 768-775; Am J Hum Genet 1999c; 64: 218-224; Transfusion 2001; 4: 276-282; Am J Obstet Gynecol 2002; 187: 1217-1221), the processed sample of blood is preferably plasma. In the following examples of the present invention, plasma obtained from the blood of pregnant women was also used for this reason.

In the present specification, "probe" means a nucleic acid or a nucleic acid analog including a base sequence complementary to a target nucleic acid sequence to be detected (ie, the Down syndrome biomarker of the present invention. do. Probes may typically be single- or double-stranded, and may be DNA as well as RNA, and may also be nucleases or heat stable peptide nucleic acid (PNA), locked nucleic acid (LNA), hexitol nucleic acid (HNA), or ANA ( It may also be a nucleic acid analog such as Altritol Nucleic Acids (MNA), Mannitol Nucleic Acids (MNA), and Ribose Nucleic Acids (RNA).

The probe is usually composed of 5 to 100 bases, but is preferably composed of 15 to 50 bases because nonspecific binding can occur if the length of the probe is too long or too short. The specific length of the probe may be determined in consideration of the conditions of use of the probe such as the temperature in the hybridization reaction, the salt concentration, the degree of complementarity in the design of the probe, and the like.

The probe can be used in combination with a label (modified covalently or labeled) to provide a signal that allows detection of hybridization within a range that does not inhibit specificity for its target nucleic acid sequence.

Such labels may be radioisotopes, fluorescent materials, chemiluminescent materials, enzymes and the like.

Specifically, the label is cyanine fluorescent dye (Cy2, Cy3, Cy5), Alexa Fluor (Alexa Fluor 350, 430, etc.), Fluorescein (fluorescein), Texas red, Flutescein isothiocyanate (FITC), Rhodamine ( rhodamine, alkaline phosphotase, horseradish peroxidase, biotin, SYTO (SYTO-11, etc.), ethidium bromide, ethidium homodimer-1, ethidium homo Dimer-2, ethidium derivative, acridine, acridine orange, acridine derivative, ethidium-acridine heterodimer, ethidium monoazide, propidium iodide, 7-aminoactinomycin D , POPO-1, TOTO-3, and the like.

Methods of binding or labeling a label to a probe can be performed using methods known in the art. Methods such as by nick translation, end-filing methods, random priming methods, chination methods (Maxam & Gilbert, Methods in Enzymology, 1986; 65: 499), Nucleic Acid Microarray and It can be carried out using the method described in the Latest PCR Method] (published by Shujunsha, Japan), and commercially available kits using such a method such as Multiprime DNA Labeling System (Amersham), DNA Labeling Kit (Takara), ARES DNA Labeling Kit (Invitrogen) etc. can be used.

The signal provided by the label can be fluorescence, radioactivity, color development, weight, X-ray diffraction or absorption, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, nanocrystals, and the like, through the signal to its target nucleic acid sequence. Can be analyzed qualitatively or quantitatively.

Specifically, the signal can be detected by various methods depending on the type of label used. For example, when an enzyme is bound to a probe as a label, the substrate of the enzyme can be reacted with the result of the hybridization reaction to detect hybridization. Combinations of such enzymes and substrates can be exemplified by chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin and the like when the enzyme is a peroxidase (e.g. horseradish peroxidase). In the case of alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) and the like can be exemplified as its substrate. For example, when the probe is labeled with gold particles, the silver nitrate may be used to detect the silver dye. If ethidium bromide is used, the probe may be detected by fluorescence detection by UV irradiation or the radiolabeled probe. In the case of can be detected by autoradiography. The detection value of the signal provided by the label can be processed statistically and quantitatively using methods known in the art, such as fluorescent image scanners and appropriate software commercially available.

The probe may be used with or added to a label, in a nucleic acid sample containing its target nucleic acid sequence, during the amplification reaction of the target nucleic acid sequence, or after the amplification reaction.

Especially when fluorophores (e.g., 6-carboxyfluorescein, tetrachlorofluorescin, etc.) and quenchers (e.g., tetramethylrhodamine, dihydrocyclopyrroloindole tripeptide minor groove binder, etc.), such as TaqMan ™ probes, are bound to the probe. Such probes can be added during amplification of the target nucleic acid sequence and used to quantify the degree of amplification in real time.

As used herein, a "nucleic acid sample" may be defined as a composition comprising a Down Syndrome marker of the invention specific for a probe of the invention, which nucleic acid sample typically contains nucleic acids that are nonspecific to the probe of the invention, in particular methylated. May comprise a PDE9A fragment. Such nucleic acid samples are obtained by using a kit for extracting nucleic acids from processed samples of pregnant woman blood such as blood of pregnant women or plasma.

As used herein, "nucleic acid" refers to double stranded or single stranded DNA or RNA, and is meant to include genomic DNA and artificial DNA synthesized using the genomic DNA as a template.

On the other hand, the hybridization condition of the probe with the target nucleic acid sequence is influenced by the length of the probe, the temperature during the hybridization reaction, salt concentration, pH, ionic strength, GC content, and the like.

Hybridization conditions include selecting a condition in which a probe can hybridize only to a nucleic acid that specifically binds to at least a nucleic acid sample that contains a nucleic acid to which a probe specifically binds to a non-specifically bound nucleic acid. desirable. By "specific binding" is meant that the probe hybridizes with the target nucleic acid sequence to form a double stranded hybrid and does not substantially form such hybrid with other nucleic acid sequences. The term "substantially" here means that nonspecific binding can be formed, which can be removed by selection of appropriate hybridization conditions, washing operations after hybridization, and the like. Detailed and appropriate conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), M. L. M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999), Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985), and the like.

On the other hand, samples of pregnant women's plasma and the like have the same sequence as the Down syndrome biomarker of the present invention but contain fragments of the methylated PDE9A gene. Therefore, when the probe of the present invention is used based on the nucleotide sequence of the down syndrome biomarker of the present invention is used, when the sample contains the methylated PDE9A gene fragment in addition to the down syndrome marker of the present invention, the probe is methylated PDE9A. Problems with specific binding to gene fragments can also occur.

To this end, it is desirable to treat the nucleic acid sample with a reagent that only modifies the cytosine of the unmethylated nucleic acid (ie without modifying the cytosine of the methylated nucleic acid), and to prepare a probe based on the sequence to be modified by the treatment of the reagent. . As such reagents, bisulfite is known that transforms cytosine of unmethylated nucleic acids into uracil. Nucleic acid modified with uracil is converted into thymine during the amplification of the nucleic acid. Thus, when using a reagent that modifies the cytosine of an unmethylated nucleic acid, the probe will be constructed with the cytosine substituted in its target nucleic acid sequence as uracil or thymine.

For the reasons mentioned above, the detection kit of Down syndrome biomarker of the present invention further comprises a reagent for modifying cytosine of unmethylated nucleic acid, in particular bisulfite, which transforms cytosine of unmethylated nucleic acid into uracil. In this case, the probe is preferably made based on the sequence to be modified by the treatment of such a reagent.

On the other hand, when the Down syndrome biomarker of the present invention is detected using a sample obtained by removing a methylated PDE9A gene fragment from a sample such as pregnant plasma or a nucleic acid sample obtained therefrom, noise (ie, background) is removed. Accurate detection results can be obtained.

Therefore, the kit of the present invention further includes a means for selectively removing methylated nucleic acid, such as a reagent for selectively removing methylated nucleic acid, or a kit for such purpose, in order to remove the methylated PDE9A gene fragment. It is desirable to.

Examples of such reagents include proteins (eg, Methyl-CpG binding domain protein2, etc.) that recognize and bind methylated CpG motifs, and enzymes (eg, McrBC, etc.) and restriction enzymes that recognize and cleave methylated CpG motifs. As the kit, EpiXplore ™ Methylated DNA Enrichment Kit (TaKaRa), SA Biosciences ™ DNA Methylation Enzyme Kit (Qiagen), and the like are commercially available.

When using such reagents or kits, the down syndrome biomarker of the invention, which is an unmethylated nucleic acid, can be selectively amplified and detected with higher accuracy.

On the other hand, samples of pregnant women's blood, plasma, etc., fetal DNA is present in a mixture with maternal mother DNA, maternal DNA contains more components than fetal DNA components. Therefore, when a nucleic acid sample is obtained from a sample of blood, plasma, etc. of a pregnant woman, the fetal DNA may be extracted and used at a higher component ratio to detect down syndrome biomarkers of the present invention. Methods of extracting higher component ratios of fetal DNA are known in the art. See, eg, the method set forth by Li Y et al. [Li Y, et al., Jama 293: 843-9 (2005); Li Y, et al., Prenat. Diagn. 24: 896-8 (2004a); Li Y, et al., Clin. Chem. 50: 1002-11], the method proposed by Dhallan et al. (JAMA. 2004; 291: 1114-1119), and the like, and kits for extracting higher component ratios of fetal DNA, such as NucliSens Magnetic Extraction System) (bioMerieux, Marcy L'Etoile), QIAamp DSP Virus Kit (QIAGEN), QIAamp Blood Mini Kit (QIAGEN), High Pure PCR Template Preparation Kit (Roche), Magna Pure LC (Roche), and the like. The meaning of "extracting higher component ratio of fetal DNA" here means that when extracting a nucleic acid sample from a processed sample of maternal blood or blood of a pregnant woman, such as plasma, the ratio of the component of fetal DNA to maternal DNA in the nucleic acid sample. This means that extraction is performed so that the ratio of the component of the fetal DNA to the maternal DNA in the processed sample of the pregnant woman's blood or the blood of the pregnant woman such as plasma is higher. It is known that fetal DNA is present in the early stages of pregnancy (week 11-17) at 3.4% (by weight) and at end-pregnancy (weeks 37-43) at 6.2% ( Am J Hum Genet 1998b; 62: 768-775; Am j hum Genet 1999c; 64: 218-224; Transfusion 2001; 4: 276-282; Am j obstet Gynecol 2002; 187: 1217-1221). Thus, for example, if a nucleic acid sample is extracted so that the fetal DNA is 7.0% of the nucleic acid sample extracted from the plasma of early pregnancy, it is a higher component ratio of the fetal DNA.

Therefore, the kit of the present invention may further include a means for extracting a nucleic acid sample from a sample such as plasma of a pregnant woman, preferably a means for extracting a higher component ratio of the fetal DNA as described above.

The kit of the present invention may also further comprise nucleic acid amplification means for amplifying Down syndrome biomarkers. Nucleic acid amplification means for amplifying Down syndrome biomarkers include DNA polymerases, nucleotides (dATP, dCTP, dGTP, dTTP, etc.) in addition to including primers specific for Down syndrome biomarkers.

A primer is a single stranded oligonucleotide that can specifically bind to template DNA and act as a starting point for DNA synthesis. Such primers may be RNA as well as DNA, and these primers may also be a combination of analogs of various types of nucleotides. Examples of nucleotide analogues include deoxyinosine nucleotides, deoxyuracil nucleotides, nucleotide analogues with modified bases such as 7-deazaguanine, nucleotide analogues with ribose analogs, and the like.

The length of the primer is not particularly limited as long as it can specifically bind to template DNA to form stable complementary bonds and serve as a starting point for DNA synthesis. The length of the primer will be determined in consideration of amplification conditions such as temperature, salt concentration of the buffer. It will usually be designed and manufactured to have a length in the range of 10 to 100 nucleotides, but since 15 to 50 nucleotides, especially 15 to 30 lengths, nonspecific binding can be formed with the template DNA if the length of the primer is too short or too long. It is desirable to be designed and manufactured to have. In addition, the sequence of the primer need not be completely complementary to the template DNA, and it is sufficient that it is complementary enough to form a specific bond with the template DNA to obtain a desired amplification product.

The primers, like the probes, may be used for the treatment of such reagents when the detection kit of the down syndrome biomarker of the present invention further contains a reagent for modifying cytosine of an unmethylated nucleic acid for the reasons described in connection with the probe. It is preferable that it is produced based on the modified sequence.

In the case of DNA polymerase, various DNA polymerases such as Klenow fragment of E. coli DNA polymerase I and thermally stable DNA polymerase can be used, but heat such as Taq DNA polymerase, Tth DNA polymerase and Pfu DNA polymerase can be used. Preference is given to using stable polymerases.

The amplification means for amplifying the down syndrome biomarker may further include a cofactor of an amplification reaction such as Mg2 +, and may further include nucleotides (Cy3-dCTP, Cy5-dCTP, etc.) labeled with fluorescent dyes. have.

In the case of using nucleotides labeled with fluorescent dyes, the amplification degree can be quantitatively and quantitatively analyzed without using the above-described probe.

Therefore, when using nucleotides labeled with fluorescent dyes, the kit of the present invention does not include a probe, and primers, DNA polymerases and labeled nucleotides (Cy3-dCTP, Cy5-dCTP, etc.) specific to the Down syndrome biomarker of the present invention. ) And a mixture of nucleotides (dATP, dCTP, dGTP, dTTP, etc.).

Amplification reactions, as well as a general PCR method, qMSP (Real-time quantitative methylation -specific polymerase chain reaction), Hot-start PCR, Nested PCR, Multiplex PCR, DOP (degenerate oligonucleotide primer) PCR, Quantitative RT (Real time) -PCR Modified PCR methods such as In-Situ PCR, Micro PCR, or Lab-on a chip PCR reaction, and isothermal amplification methods such as rolling circle amplification (RCA) may be used.

The kit for detecting Down syndrome marker of the present invention may further comprise a probe or primer specific for the gene fragment for detecting the methylated PDE9A gene fragment.

The methylation if PDE9A detected and amplified at the same time, a gene fragment with Down syndrome markers of the present invention, in order to rule out the specificity for the specific probe or primer is Down syndrome biomarkers of the present invention the methylation PDE9A gene fragment, the methylation PDE9A gene fragment The design of the probe or primer is preferably made based on the base sequence to be modified by treatment of a reagent for modifying cytosine of an unmethylated nucleic acid for the reason described above. Therefore, in this case, the kit of Down Syndrome Biomarker of the present invention preferably further includes a reagent for modifying cytosine of unmethylated nucleic acid, particularly bisulfite for modifying cytosine of unmethylated nucleic acid to uracil.

The methylated PDE9A gene fragment may be detected and amplified separately. Such detection and amplification is performed by processing a protein that recognizes and binds a methylated CpG motif (eg, Methyl-CpG binding domain protein2, etc.) to isolate a methylated nucleic acid sample containing a methylated PDE9A gene fragment, followed by amplification and detection procedures. Will be done through.

The value determined for the methylated PDE9A gene fragment contained in the processed sample, such as pregnant blood, or plasma, is combined with the value determined for the Down syndrome biomarker of the present invention contained in the processed sample of the pregnant woman, such as blood, or plasma of the pregnant woman. It can be more useful for the diagnosis of pregnant women with Down syndrome.

As confirmed in the Examples of the present invention below, all four combinations, including the non-methylation index, are more accurate than the accuracy (AUC) based on the value determined for the concentration of Down Syndrome marker of the present invention.

In the present specification, the combined value of the concentration in the non-methylated PDE9A blood, which is the Down syndrome biomarker of the present invention, and the concentration in the blood of the methylated PDE9A is determined by [(value determined for the concentration of the Down syndrome biomarker) / (the concentration of the Down syndrome biomarker). Value determined for the concentration of methylated PDE9A gene fragment), [(value determined for the concentration of methylated PDE9A gene fragment) / (value determined for the concentration of Down syndrome biomarker + concentration of methylated PDE9A gene fragment) Value determined for the concentration of Down's syndrome marker / (value determined for the concentration of methylated PDE9A gene fragment)], or [(value determined for the concentration of methylated PDE9A gene fragment) / (Down syndrome) Value determined for the concentration of the biomarker).

As used herein, "methylated PDE9A gene fragment" refers to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 as some sequences in the PDE9A gene. Preferably it means a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.

Meanwhile, the kit of the present invention may further include a standard nucleic acid calibrator for the Down syndrome marker of the present invention. Standard nucleic acid sequences for calibration curves include sequences that specifically bind to specific probes for the Down Syndrome biomarkers of the invention as described above, and specific primers for the Down Syndrome biomarkers of the invention as described above. It is preferable to make it. More preferably, the probes and / or primers of the present invention comprise the same sequence as that of the region of the Down syndrome marker of the present invention to which the probe and / or primer of the present invention bind.

These standard nucleic acid sequences are prepared at various concentrations and amplified under the same amplification conditions as the Down syndrome biomarker of the present invention, and for the purpose of calibrating the concentration in the blood of the Down syndrome biomarker of the present invention by quantifying the degree of amplification versus concentration. Used.

The kits of the invention may also further comprise standard nucleic acid sequences for the methylated PDE9A gene fragment. The standard nucleic acid sequence also methylated PDE9A specific probe and specific primers and and specific seoyeolreul preferably comprises a coupling, methylation of combining a specific probe and primer for methylation PDE9A gene fragment for the gene fragment PDE9A of gene fragments More preferably, it comprises the same sequence as the sequence of the region.

In another aspect, the method comprises the steps of: (a) obtaining blood or a processed sample of blood from a pregnant woman, and (b) determining the concentration of Down syndrome biomarker in the pregnant blood or processed sample of the blood. It can be grasped by the detection method of syndrome baromarker.

In the method of the present invention, the processed sample of blood of step (a) is plasma or serum, preferably plasma, for the reasons described above.

In addition, in the method of the present invention, the pregnant woman in the step (a) is preferably a pregnant woman in the first half of pregnancy. Here, the first trimester is from 5 weeks of gestation to 20 weeks of gestation capable of detecting Down syndrome biomarker of the present invention, preferably from 5 to 12 weeks as in the following examples.

In the method of the present invention, the step of determining the concentration of Down syndrome biomarker in the maternal blood of step (b), or the processed sample of the blood, preferably (b-1) the maternal blood, or processed sample of the blood And (b-2) determining the concentration of Down syndrome biomarker in the extracted nucleic acid sample.

Extracting the nucleic acid sample from the maternal blood of the step (b-1), or the processed sample of the blood, as described above, which can extract fetal DNA at a higher component ratio, the method of Li Y. et al., Dhallan It is preferable to carry out using the method proposed by et al. Or a commercially available kit for this purpose.

In addition, the step of extracting the nucleic acid sample from the maternal blood of the step (b-1) or the processed sample of the blood is preferably performed after removing the methylated PDE9A gene fragment contained in the maternal blood or the processed sample of the blood. . Removal of the methylated PDE9A gene fragment can be performed using a restriction enzyme that recognizes and cleaves a methylated CpG motif or a protein that recognizes and binds a methylated CpG motif as described above, or using a commercially available kit for this purpose.

Determining the concentration of the down syndrome biomarker in the extracted nucleic acid sample of step (b-2) (b-2-1) amplifying the down syndrome biomarker in the extracted nucleic acid sample, (b-2-2) Determining the concentration of the amplified Down syndrome biomarker.

Amplifying the down syndrome biomarker in the nucleic acid sample may be performed using a specific probe, primer, DNA polymerase, and the like for the down syndrome marker of the present invention as described above.

The step of determining the concentration of the amplified Down syndrome biomarker is to measure the signal of the label using a label bound or labeled probe or a label with the probe, or if necessary in the same amplification conditions Measurements can be made using calibration curves prepared using amplified standard nucleic acid samples.

In the method of the invention, the step of determining the concentration of methylated PDE9A in pregnant blood, or processed sample of the blood, may further be included.

As described above, the value determined for the concentration of methylated PDE9A may be converted into a combination value, such as arithmetic operation and the value determined for the concentration of Down syndrome marker of the present invention, which may be more useful for diagnosing pregnant women with Down syndrome. Because there is.

In another aspect, the invention relates to a method for providing information useful for diagnosing Down syndrome.

A method for providing useful information for diagnosing Down syndrome of the present invention comprises the steps of (a) obtaining blood or processed samples of blood from a pregnant woman, (b) concentration of Down syndrome biomarker in pregnant blood or processed samples of the blood. (C) determining the concentration of methylated PDE9A gene fragment in maternal blood, or processed sample of the blood, and (d) determining the concentration of Down syndrome biomarker in step (b). Combining the values determined for the concentration of the methylated PDE9A gene fragment of step (c) comprises the step of obtaining a combined value.

In the method, the combined value is [(value determined for the concentration of Down syndrome biomarker) / (value determined for the concentration of Down syndrome biomarker + value determined for the concentration of methylated PDE9A gene fragment)], [( Value determined for the concentration of the methylated PDE9A gene fragment) / (value determined for the concentration of the Down syndrome biomarker + value determined for the concentration of the methylated PDE9A gene fragment)], [(value determined for the concentration of the Down syndrome marker) / ( Value determined for the concentration of the methylated PDE9A gene fragment)], or [(value determined for the concentration of the methylated PDE9A gene fragment) / (value determined for the concentration of Down syndrome biomarker)].

In another aspect, the invention relates to a method for diagnosing Down syndrome. As for the down syndrome diagnosis method of the present invention, what has been described above in connection with the detection method of the down syndrome biomarker of the present invention is effective as it is.

In another aspect, the present invention relates to the use of a primer or probe as described above for the manufacture of a kit for detecting down syndrome biomarker.

In a kit, method, or the like for detecting the Down syndrome biomarker of the present invention in a preferred embodiment, the probe or primer specific for the Down syndrome biomarker of the present invention is positively selected from the probe (SEQ ID NO: 3) used in the examples below. Reverse primers (SEQ ID NOs: 4 and 5, respectively). If the methylated PDE9A gene fragment needs to be detected, the probes and primers specific for the methylated PDE9A gene fragment are also the probes (SEQ ID NO: 6) and the forward and reverse primers used in the examples below (SEQ ID NO: 7 and 8). These probe and primer sequences are designed and constructed based on sequences to be modified by treatment of bisulfite, which transforms cytosine of unmethylated nucleic acids into uracil.

As described above, the present invention can provide a detection kit and a detection method of Down syndrome biomarker. In addition, the present invention can provide a method for diagnosing Down syndrome.

The down syndrome biomarker detection method of the present invention can be used as a non-invasive prenatal diagnosis method and can be applied irrespective of the sex of the fetus, and has high accuracy (AUC), specificity, sensitivity, etc. It is expected to be able to replace the noninvasive diagnosis method of syndrome.

1 is unmethylated PDE9A concentration in the blood ( "UPDE"), unmethylated index ( "UMI"), and a ROC curve of [unmethylated PDE9A concentration in the blood / methylated PDE9A concentration in the blood] X1,000 ( "U_M") The result of the analysis.
FIG. 2 shows the concentration in methylated PDE9A blood (“MPDE”), [(concentration in methylated PDE9A blood) / (concentration in methylated PDE9A blood + concentration in unmethylated PDE9A blood)] × 1,000 (“MI”), and [methylated] Concentration in PDE9A blood / unmethylated concentration in PDE9A blood] X1,000 (“M_U”).

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

< Example > Down syndrome Biomarker  Experimental Methods and Experimental Results for Detection

< Example  1> Experiment method

< Example  1-1> How to obtain maternal plasma DNA  Extraction method

Maternal blood was obtained from women recruited for noninvasive prenatal diagnostic studies at Cheil Hospital (Seoul, Korea). All participants were prenatal care at Cheil General Hospital, and only single-pregnant women were selected during the first trimester (5-12 weeks). Ultrasonography prior to early pregnancy blood sampling was performed to confirm viability of singlet pregnancy and the number of weeks of pregnancy. Prior to the start of the study, the number of samples in the experimental and control groups was determined by priori analysis of the G * Power program (Heinrich-Heine-Universitat, Germany). Eighteen experimental groups and ninety control groups provided at least 85% statistical power to confirm the difference between the experimental and control groups in a two-sided test with an effective size of 0.8, an allocation ratio of 5, and an α error of 0.05. Eighteen women were identified as pregnant with Down's syndrome fetus by amniotic fluid, chorionic villus biopsy, and karyotyping of aborted tissue. The control group was selected from 90 women who delivered healthy normal infants at the end of pregnancy without medical or obstetric complications. Each experimental case was paired with five control cases that matched the storage period of the sample and the number of weeks of gestation in the blood sample. This study was approved by the Medical Research Review Board of Cheil General Hospital (# CGH-IRB-2008-43) with the written consent of the participants.

Maternal blood was immediately centrifuged at 1,600 g for 10 minutes immediately after acquisition to obtain only the plasma portion, and the obtained plasma portion was subjected to further centrifugation at 16,000 g for 10 minutes to take pure plasma except for sediment. Maternal plasma DNA was extracted using a QIAamp DSP Virus Kit (Qiagen), which can be extracted at 0.5 mL plasma with a higher component ratio than fetal DNA.

< Example  1-2> methyl  Specific qMSPCR ( quantitative methylation - specific  중합체 chain reaction )

DNA extracted from maternal plasma induced sequence changes of unmethylated DNA moieties by bisulfite, which transforms cytosine of unmethylated nucleic acids into uracil using the EZ DNA Methylation Kit (Zymo Research).

The mixed solution to be used for PCR consists of 5 uL of DNA denatured by bisulfite, 12.5 uL of 2X IQ Supermix kit (Bio-Rad) at a final volume of 25 uL, and 200 nM each of primers and probes.

The primer and probe sequences used for PCR of methyl PDE9A (hereinafter "M-PDE9A") and nonmethyl PDE9A (hereinafter "U-PDE9A") are as follows.

Probe of U-PDE9A: 5'-FAM-TTT GTT TGG TGA TGT TAT GTG GTT T-MGBNFQ-3 '(SEQ ID NO: 3), Forward primer: 5'-GGT TTG TTT TGG TGA GTG TGT GTC GT-3' ( SEQ ID NO: 4), Reverse primer: 5'-CCC AAC CAT CCC AAA AAA GCA-3 '(SEQ ID NO: 5)

Probe of M-PDE9A: 5'-FAM-TTC GGT GAC GTT ACG CGG T-MGBNFQ-3 '(SEQ ID NO: 6), forward primer: 5'-CGG TGA GTG CGC GTC GC-3' (SEQ ID NO: 7), Reverse primer: 5'-CCA ACC ATC CCG AAA AAG CG-3 '(SEQ ID NO: 8)

The probe was bilaterally labeled with 6-carboxyfluorescein (FAM) and minor groove-binding non-fluorescent quencher (MGBNFQ).

PCR conditions were repeated 10 times at 95 ℃ for 15 seconds at 95 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ was repeated a total of 50 times. The concentrations of M-PDE9A and U-PDE9A in blood were analyzed using calibration curves using serially diluted M-PDE9A and U-PDE9A standard nucleic acid calibrators. The standard nucleic acid sequences used are as follows: M-PDE9A is 5'-CGG TGA GTG CGC GTT GCG GGT TTT GTT CGG TGA CGT TAC GCG GTT TTT TCG TTT TTT CGG GAT GGT TGG-3 '(SEQ ID NO: 9), U -PDE9A is 5'-GGT TTG TTT TGG TGA GTG TGT GTT GTG GGT TTT GTT TGG TGA TGT TAT GTG GTT TTT TTG TTT TTT TGG GAT GGT TGG G-3 '(SEQ ID NO: 10).

Concentrations in M-PDE9A and U-PDE9A in blood were expressed in genome equivalent (GE) / mL based on previous reports ( Clin Chem . 2001; 47: 1607-1613, Am J Hum Genet . 1998; 62: 768-775). Each assay included a negative control with water. All experiments were repeated three times, and the inter- and intra-assay coefficients of variation for this method were 5.9% and 7.2% for M-PDE9A, respectively. The cases were 6.4% and 8.1%, respectively. The final data represents the average of the results from repeated experiments.

< Example  1-3> Sequencing

After the PCR product was purified using a PCR purification kit, 20ul of PCR reaction solution was prepared using 20ng of purified PCR product, 3.5ul of 5X Sequencing Buffer, Bigul 1ye, DW 14ul, and 0.5ul of primer. PCR reaction conditions were performed 25 times amplification reaction for 96 minutes 10 seconds, 50 ℃ 5 seconds, 60 ℃ 4 minutes, and then mixed with 100 mM EDTA 12.5uL, DW 17.5uL, 100% EtOH and 10 minutes at room temperature. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was removed, and 200% of 70% EtOH was added thereto, followed by washing. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was removed. After drying at 50 ° C, 10 μL of Formamide was dispensed, and the nucleotide sequence was analyzed using ABI PRISM 3100 Genetic Analyzer.

< Example  1-4> Statistical analysis

Clinical characteristics of the experimental and control groups were analyzed using chi test (χ ² -test) and Mann-Whitey U test. Amounts in the blood of M-PDE9A and U-PDE9A were expressed in medians (quartile) and analyzed between experimental and control groups using the Mann-Whitey U test.

The accuracy of fetal down syndrome detection was analyzed with concentrations in the U-PDE9A blood and unmethylation index, and determined with fetal karyotype analysis. The demethylation index here was calculated as [(concentration in U-PDE9A blood) / (concentration in M-PDE9A blood + concentration in U-PDE9A blood)] × 1,000.

ROC curve analysis was performed to determine the optimal cut value, and the cut value was selected as a 5% false positive rate to compare with the diagnostic accuracy of current Down syndrome screening methods.

Sensitivity, positive predictive value, negative predictive value, and risk rate for assessing diagnostic accuracy using U-PDE9A blood concentration and non-methylation index are measured using the EpiMax Table Calculator (http://www.healthstrategy.com/epiperl/epiperl.htm). Calculated and compared under the same 5% false positive rate.

The accuracy of the overall diagnostic method was assessed with the area under the ROC curve (AUC). The overall diagnostic accuracy using the area under the ROC curve is in addition to the concentrations in U-PDE9A blood and unmethylation index, as well as other combinations of concentrations in U-PDE9A blood and concentrations in M-PDE9A and M-PDE9A. Calculated. Where another combination of the concentration in U-PDE9A blood and the concentration in M-PDE9A blood is [(concentration in M-PDE9A blood) / (concentration in M-PDE9A blood + concentration in U-PDE9A blood)] X1,000, [(Concentration in M-PDE9A blood) / (concentration in U-PDE9A blood)] × 1,000, and [(concentration in U-PDE9A blood) / (concentration in M-PDE9A blood) × 1,000.

ROC curve analysis was performed to determine the optimal cut value, and the cut value was selected as a 5% false positive rate to compare with the diagnostic accuracy of current Down syndrome screening methods.

Sensitivity, positive predictive value, negative predictive value, and risk rate for assessing diagnostic accuracy using U-PDE9A blood concentration and non-methylation index are measured using the EpiMax Table Calculator (http://www.healthstrategy.com/epiperl/epiperl.htm). Calculated and compared under the same 5% false positive rate.

The accuracy of the overall diagnostic method was assessed with the area under the ROC curve (AUC).

Logistic regression analysis was used to assess the amount and demethylation index of U-PDE9A blood as a risk factor for fetal down syndrome, and maternal age at the time of blood collection as a potential confounding factor. It became. The risk for which the potential confounding variable was corrected was compared with the uncorrected risk. Statistical analysis was done using Statistical Package for Social Sciences 10.0 (SPSS Inc.), and statistical significance was set to P <0.05 in all tests.

< Example  2> Experiment result

< Example  2-1> M- PDE9A And U- PDE9A  Sequencing results

The amplified sequences of each of M-PDE9A and U-PDE9A are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. SEQ ID NO: 11 and SEQ ID NO: 12 of the amplified sequence of the sequence changed by bisulfite (unmethylated cytosine changed to thymine) and SEQ ID NO: 1 (the sequence on the genome of M-PDE9A) and SEQ ID NO: 2 (U- Compared to the genomic sequence of PDE9A), it is possible to determine which cytosine of the sequences of M-PDE9A and U-PDE9A is methylated. In addition, the amplified sequence of each of the M-PDE9A and U-PDE9A can be confirmed to match the standard nucleic acid sequence of each of M-PDE9A and U-PDE9A.

< Example  2-2> Assessment of Down Syndrome Diagnostic Scale

At the time of blood collection, the mother's age was significantly higher in the experimental group than in the control group ( P = 0.016). However, body mass index was not different between the control and experimental groups ( P = 0.198). Median gestational age was 7.5 weeks in both control and experimental groups. The frequency of maternal mothers, fetal sex and smoking status did not differ between the two groups.

In the analysis of M-PDE9A and U-PDE9A by fetal gender, there was no difference in fetal gender. In analysis of M-PDE9A and U-PDE9A according to fetal karyotype, M-PDE9A did not differ between control and experimental groups [1743.1 (1613.5-2446.3) vs 2023.4 (1573.0-2564.0) GE / mL, P = 0.520]. However, U-PDE9A was significantly higher in the experimental group than the control group [567.5 (422.3-693.3) vs 163.5 (95.6-271.4) GE / mL, P <0.001].

For evaluating the diagnostic accuracy of U-PDE9A and non-methylation index, the cleavage of each factor was selected at 5% false positive rate by ROC analysis and compared sensitivity, positive predictive value, negative predictive value and AUC. Cleavage of U-PDE9A was 415 GE / mL with 77.8% sensitivity, 73.7% positive predictive value, 95.5% negative predictive value, and 0.941 AUC (95% confidence interval: 0.889-0.992). Cleavage, sensitivity, positive predictive value, negative predictive value, and AUC of unmethylated index were 140, 83.3%, 75.0%, 96.6%, and 0.970 (95% confidence interval: 0.938-1.002), respectively.

Analysis of the association between U-PDE9A and non-methylation indices in the risk of pregnancy with Down syndrome fetuses significantly increased the risk of Down syndrome in women with higher cuts than U-PDE9A and non-methylation indices. 59.5-fold increase for U-PDE9A, 85.0-fold increase for non-methylation index). Moreover, the risk of U-PDE9A and non-methylation index was increased by 56.8 and 73.5 times, even if the mother's age was corrected. Therefore, women with U-PDE9A and non-methylation indices above cuts had a significantly increased risk of Fetal Down syndrome than women with cuts below cuts.

Finally, the diagnostic accuracy (AUC) of M-PDE9A is 0.548, [(concentration in M-PDE9A blood) / (concentration in M-PDE9A blood + concentration in U-PDE9A blood)] X1,000 is 0.970, [(M- Concentration in PDE9A Blood / Concentration in U-PDE9A Blood)] X1,000 was 0.970, [(Concentration in U-PDE9A Blood) / (Concentration in M-PDE9A Blood)] X1,000 was 0.969.

Results of the analysis of the respective ROC curves of the combination of the concentrations in U-PDE9A blood and the concentrations in the blood of M-PDE9A other than U-PDE9A, M-PDE9A, unmethylated index, and unmethylated index are shown in FIGS. 2 is disclosed. 1 and 2, U-PDE9A is "UPDE", M-PDE9A is "MPDE", demethylation index is "UMI", [(concentration in M-PDE9A blood) / (concentration in M-PDE9A blood) + Concentration in U-PDE9A Blood)] X1,000 is "MI", [(Concentration in M-PDE9A Blood) / (Concentration in U-PDE9A Blood] X1,000 is "M_U", [(U-PDE9A Concentration in blood) / (concentration in M-PDE9A blood] X1,000 is indicated as "U_M".

In FIG. 1, the unmethylation index and the ROC curve of [(amount in U-PDE9A blood) / (amount in M-PDE9A blood)] × 1,000 are shown as one curve, and in FIG. 2, [(M -Amount in PDE9A blood) / (amount in M-PDE9A blood + amount in U-PDE9A blood)] X1,000 and [(amount in M-PDE9A blood) / (amount in U-PDE9A blood)] X1,000 It is also shown as a curve because it is almost identical.

Attach an electronic file to a sequence list

Claims (25)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete (a) determining the concentration of unmethylated PDE9A gene fragment in the blood of a pregnant woman or a processed sample of the blood,
(b) determining the concentration of the pregnant woman's methylated PDE9A gene fragment in the pregnant woman's blood or processed sample of the blood, and
(c) a step to obtain the combined value by combining the determined values for the concentration of methylated PDE9A gene fragment of the determined values and the step (b) for the unmethylated PDE9A concentration of the gene fragment of step (a) pregnant women Composed of,
The combined values are [(value determined for concentration of unmethylated PDE9A gene fragment) / (value determined for non-methylated PDE9A gene fragment + value determined for concentration of methylated PDE9A gene fragment)], [(of methylated PDE9A gene fragment) Value determined for concentration) / (value determined for concentration of unmethylated PDE9A gene fragment + value determined for concentration of methylated PDE9A gene fragment)], [(value determined for concentration of unmethylated PDE9A gene fragment / (methylated PDE9A) Value determined for the concentration of the gene fragment)], or [(value determined for the concentration of the methylated PDE9A gene fragment) / (value determined for the concentration of the unmethylated PDE9A gene fragment)]. How to provide information.
25. The method of claim 24,
Method for providing useful information for the diagnosis of Down syndrome, characterized in that the processed sample of the pregnant woman blood is plasma.

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