BRPI0711273A2 - methods for detecting or monitoring a disorder associated with pregnancy, and for detecting trisomy 21 in a fetus in a pregnant woman - Google Patents

Abstract

METHODS TO DETECT OR MONITOR A DISTURBANCE ASSOCIATED WITH PREGNANCY, AND TO DETECT TRISOMY 21 IN A FETUS IN A PREGNANT WOMAN. This application provides the use of new fetal markers for diagnosis and prenatal monitoring of certain conditions related to pregnancy. More specifically, the invention lies in the discovery that certain CpG islands located on the fetal chromosome 21 demonstrate a methylation profile that is distinct from that of the corresponding CpG islands located on the maternal chromosome 21. This application also provides kits for diagnosing or monitoring relevant conditions .

Description

"METHODS FOR DETECTION OR MONITORING A PREGNANCY ASSOCIATED DISORDER, AND FOR DETECTING TRISSOMY 21 IN A FETUS IN A PREGNANT WOMAN"

RELATED ORDERS

This application claims priority for U.S. Provisional Patent Application No. 60 / 797,456, filed May 3, 2006, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Early detection of pregnancy-related conditions, including potential complications during pregnancy or childbirth, and genetic defects of the fetus is of crucial importance, as well as allowing early medical intervention necessary for the safety of both mother and fetus. Prenatal diagnosis is routinely conducted using cells isolated from the fetus through procedures such as villous chorion sampling (CVS) or amniocentesis. These conventional methods, however, are invasive and present an appreciable risk to both mother and fetus despite more careful manipulation (Tabor et al., Lancet 1: 1287-1293, 1986).

Alternatives to these invasive methods have been developed for prenatal evaluation, for example, to detect fetal abnormalities, following the findings that various types of fetal cells can be found in the maternal circulation (Johansen et al., Prenat. Diagn. 15 : 921-931, 1995) and more importantly, circulating cell-free fetal DNA can be detected in plasma and maternal serum (Lo et al., Lancet 350: 485-487, 1997). The amount of fetal DNA in maternal blood has been shown to be sufficient for genetic analysis without complex plasma or serum treatment, in contrast to alternative methods that require steps for fetal cell isolation and enrichment. Genotyping of fetal rhesus D (RhD) (Lo et al., N Engl. J Med. 339: 1734-1738, 1998), Sex determination of the fetus (Costa et al., N Engl. J. Med. 346: 1502 , 2002) and diagnoses of various fetal disorders (Amicucci et al, Clin. Chem. 46: 301-302, 2000; Saito et al., Lancet 356: 1170, 2000; and Chiu et al., Lancet 360: 998-1000 , 2002) were performed by detecting fetal DNA in maternal plasma or serum using a polymerase chain reaction (PCR) based technique.

In addition, quantitative fetal DNA abnormalities in plasma / maternal serum were presented in preeclampsia (Lo et al, Clin. Chem. 45: 184-188, 1999 and Zhong et al., Am. J Obstet. Gynecol. 184 : 414-419, 2001), fetal trisomy 21 (Lo et al, Clin. Chem. 45: 1747-1751, 1999 and Zhong et al, Prenat. Diagn. 20: 795798, 2000) and gravid hyperemesis (Sekizawa et al. Clin Chem 47: 2164-2165, 2001). Detection of fetal nucleic acid in maternal blood for prenatal genetic analysis is also disclosed in U.S. Patent No. 6,258,540.

Fetal RNA present in maternal blood has also been established as a diagnostic tool for conditions associated with pregnancy. For example, U.S. Patent Application No. 09 / 876,005 discloses noninvasive techniques based on detection of fetal RNA in maternal blood; US Patent Application No. 10 / 759,783 further discloses that the amount of certain mRNA species (e.g., β-hCG, hCRH, hPL, KISS1, TPFI2 and PLAC1) present in maternal blood can be used as markers to diagnose, monitor or predict pregnancy-related disorders such as preeclampsia, fetal chromosomal aneuploidy, and premature delivery.

Although DNA stability provides an advantage over fetal DNA-based diagnosis, there is a major limitation to this method: Both fetal and maternal DNA are present in the acellular portion of a pregnant woman's blood, for example, serum or plasma. Thus, there is a need to distinguish fetal DNA from maternal DNA to ensure accurate diagnosis. This was first disclosed in U.S. Patent Application No. 09 / 944,951, published 20030044388, such fetal and maternal DNAs can be distinguished by their different methylation profiles. Landes et al. U.S. Patent Application Publication No. 20030211522 also proposed that differential methylation markers may be used for prenatal diagnosis. In the present disclosure, various human genomic DNA sequences located on chromosome 21 are first identified as regions containing differentially methylated sites in genomic DNA originating from a fetus or an adult (for example, a pregnant woman). Thus, these differentially methylated genomic sites allow the proper identification or quantification of fetal and maternal DNA and thus reliable diagnoses of prenatal conditions.

BRIEF SUMMARY OF THE INVENTION

In the first aspect of this invention, a method is provided for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus. The method comprises the following steps: (a) obtaining a biological sample from the woman, wherein the sample is whole blood, serum, plasma, urine or saliva; (b) determine the methylation status of a genomic sequence containing CpG in the sample, in which the genomic sequence of the fetus and the female genomic sequence are differentially methylated, thereby distinguishing between the female genomic sequence and the female genomic sequence. fetus in the sample, wherein the genomic sequence is at least 15 nucleotides in length, comprising at least one cytosine, and within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit pseudogene 2 (inhibitor) (PPP1R2P2), FemlA {Caenorhabditis elegans), CGI009, carbonyl reductase I (CBR1) molecule, Down Syndrome (DSCAM) cell adhesion and open reading matrix 29 of chromosome 21 (C2 lorf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; (c) determine the level of the genomic sequence of the fetus; and (d) compare the level of the fetal genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

In some embodiments, the female genomic sequence is methylated and the fetal genomic sequence is not methylated. In other embodiments, the female genomic sequence is not methylated and the fetal genomic sequence is methylated.

In some embodiments, step (b) is performed by treating the sample with a reagent that differentially modifies methylated and unmethylated DNAs. For example, the reagent may comprise bisulfite; or the reagent may comprise one or more enzymes which preferably cleave methylated DNA; or the reagent may comprise one or more enzymes which preferably cleave unmethylated DNA. In some embodiments, step (b) is performed by methylation specific PCR.

In the second aspect of this invention, a method is provided for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus. The method comprises the steps of: (a) obtaining DNA from a woman's biological sample, where the sample is whole blood, serum, plasma, urine or saliva; (b) treating the DNA of step (a) with bisulfite; and (c) performing an amplification reaction using the DNA from step (b) and two primers to amplify a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit 2 pseudogene 2 (inhibitor) (PPP1R2P2), Similarity to FemlA (Caenorhabditis elegans), CGI009, Carbonyl Reductase 1 (CBR1), Down Syndrome Cell Adhesion Molecule (DSCAM), Chromosome 21 Open Reading Matrix (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and wherein at least one of the two primers differentially binds to the fetal genomic sequence; and (d) comparing the level of the amplified portion of the genomic sequence from step (c) to a standard control, wherein an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

In some embodiments, the amplification reaction is a polymerase chain reaction (PCR), such as a methylation-specific PCR. In other embodiments, the amplification reaction is an amplification based on the nucleic acid sequence, a strand displacement reaction, or a branched DNA amplification reaction.

This method, as well as the method described in the first aspect of this invention, is suitable for detecting or monitoring conditions such as preeclampsia, premature delivery, hyperemesis gravidarum, ectopic pregnancy, a chromosomal aneuploidy (eg trisomy 21) and delay of intrauterine growth.

In the third aspect of this invention, a method is provided for detecting and monitoring a disorder associated with pregnancy. The method comprises the steps of: (a) obtaining DNA from a woman's biological sample, where the sample is whole blood, serum, plasma, urine or saliva; (b) treating the DNA of step (a) with a reagent that differentially modifies methylated and unmethylated DNAs; (c) determining the nucleotide sequence of a CpG-containing genomic sequence from step (b), wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9 A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit 2 (PPP1R2P2) subunit (PPP1R2P2), Similarity to FemlA ( Caenorhabditis elegans), CGI009, Carbonyl Reductase 1 (CBRI), Down Syndrome Cell Adhesion Molecule (DSCAM), Chromosome 21 Open Reading Matrix 29 (C21orf29), Holocarboxylase Synthetase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) comparing the nucleotide sequence profile of step (c) with a standard control, wherein a change in the control pattern profile indicates the presence or progression of a disorder associated with pregnancy.

In some embodiments, the reagent comprises bisulfite; or the reagent may comprise one or more enzymes which preferably cleave methylated DNA; or the reagent may comprise one or more enzymes which preferably cleave unmethylated DNA.

In some embodiments, the method may further comprise an amplification step using the DNA from step (b) and two primers to amplify the genomic sequence. For example, the amplification step may be performed by PCR, such as methylation specific PCR. In some embodiments, step (c) is performed by mass spectrometry. In other embodiments, step (c) is performed by primer extension. Other possible means for performing step (c) include polynucleotide hybridization by real time PCR and electrophoresis.

In the fourth aspect of this invention, a method is provided for detecting trisomy 21 in a fetus in a pregnant woman. The method comprises the steps of: (a) obtaining a biological sample from the woman, wherein the sample is whole blood, serum, plasma, urine or saliva; (b) treating the sample from step (a) with a reagent that differentially modifies the methylated and unmethylated DNAs; (c) analyzing alleles of a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit pseudogene 2 (PPP1R2P2), FemlA (<Caenorhabditis elegans), CGI009, carbonyl reductase I (CBR1), Down Syndrome (DSCAM) cell adhesion molecule, chromosome 21 open reading matrix 29 (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) determining the ratio of alleles, wherein a deviation from that of a woman carrying a fetus having no trisomy 21 indicates trisomy 21 in the fetus.

In some embodiments, the reagent comprises bisulfite. In other embodiments, the reagent comprises one or more enzymes that preferably cleave methylated DNA. Alternatively, the reagent may comprise one or more enzymes which preferably cleave unmethylated DNA.

In some embodiments, the method further comprises an amplification step following step (b) for amplifying the methylated and unmethylated genomic sequence. The amplification step can be performed by PCR, such as methylation specific PCR.

There are several possibilities to perform step (c) of the claimed method. For example, the step may be performed by mass spectrometry, primer extension assay, real-time PCR, polynucleotide hybridization or electrophoresis. In some embodiments of this method, the two different alleles of the genomic sequence on chromosome 21 of the fetus comprise a single nucleotide polymorphism, an insert-deletion polymorphism, or a single tandem repeat polymorphism.

In the fifth aspect of this invention, a method is provided for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus. The method comprises the steps of: (a) obtaining a biological sample from the woman, wherein the sample is whole blood, serum, plasma, urine or saliva; (b) determining the level of a CpG-containing genomic sequence in the sample, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one unmethylated cytosine, and is within a region on chromosome 21 and wherein the region consists of of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit 2 pseudogene 2 (PPP1R2P2) and FemlA (Caenorhabditis elegans) Similarity , and (2) a DNA sequence of no more than 10 kb upstream and / or downstream of the site; and (c) comparing the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

In some embodiments, step (b) comprises treating DNA present in the blood sample with a reagent that differentially modifies methylated and unmethylated cytosine. This reagent may comprise bisulfite or may comprise one or more enzymes which preferably cleave DNA comprising methylated cytosine or may comprise one or more enzymes which preferably cleave DNA comprising unmethylated cytosine.

In some embodiments, step (b) comprises an amplification reaction, such as a polymerase chain reaction (PCR), especially a methylation-specific PCR. The amplification reaction may also be a nucleic acid sequence based amplification, a strand displacement reaction, a branched DNA amplification reaction. In some embodiments, the level of the genomic DNA sequence is determined by electrophoresis or polynucleotide hybridization.

The method is suitable for detecting or monitoring various disorders associated with pregnancy, including preeclampsia, premature birth, hyperemesis gravidarum, ectopic pregnancy, trisomy 21 and intrauterine growth retardation.

In the sixth aspect of this invention, a method for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus. The method comprises the steps of: (a) obtaining a biological sample from the woman, wherein the sample is whole blood, serum, plasma, urine or saliva; (b) determining the level of a CpG-containing genomic sequence in the sample, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one methylated cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI009, carbonyl reductase 1 (CBR1), Down Syndrome cell adhesion molecule (DSCAM), chromosome 21 open reading matrix (C21off29), Holocarboxylase Synthase (HLCS) and CGI132 and (2) a DNA sequence of no more than 10 kb upstream and / or downstream of the site; and (c) compare the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

In some embodiments, step (b) comprises treating the DNA present in the blood sample with a reagent that differentially modifies methylated and unmethylated cytosine. This reagent may comprise bisulfate or may comprise one or more enzymes which preferably cleave DNA comprising methylated cytosine or may comprise one or more enzymes which preferably cleave DNA comprising unmethylated cytosine.

In some embodiments, step (b) comprises an amplification reaction, such as a polymerase chain reaction (PCR), especially a methylation-specific PCR. The amplification reaction can also be a nucleic acid sequence based amplification, a strand displacement reaction, a branched DNA amplification reaction. In some embodiments, the level of the genomic DNA sequence is determined by electrophoresis or polynucleotide hybridization.

The method is suitable for detecting or monitoring various disorders associated with pregnancy, including preeclampsia, premature birth, hyperemesis gravidarum, ectopic pregnancy, trisomy 21 and intrauterine growth retardation.

In the practice of the present invention within all aspects mentioned above, a CpG island may be used as the CpG containing genomic sequence in some cases whereas in other cases the CpG containing genomic sequence cannot be a CpG island.

The methods of the invention, in all respects, can be performed without any step performed on the woman. In such embodiments, the methods of the invention omit the step of obtaining the woman's biological sample and begin with the first reported step that is performed on the sample. All other disclosures in this application apply equally to such embodiments except where the context clearly requires otherwise.

Thus, the first aspect method in this embodiment becomes: a method of detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) [omitted]; (b) determine the methylation status of a CpG-containing genomic sequence in a woman's biological sample, where the sample is whole blood, serum, plasma, urine or saliva, where the genomic sequence of the fetus and the woman's genomic sequence are differentially methylated, thus distinguishing between the female genomic sequence and the fetal genomic sequence in the sample, where the genomic sequence is at least 15 nucleotides in length, comprising at least one cytosine and is within a region on the chromosome. 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit 2 pseudogene 2 (PPP 1R2P2) , Similarity to FemlA (Caenorhabditis elegans), CGI009, Carbonyl Reductase 1 (CBR1), Down Syndrome Cell Adhesion Molecule (DSCAM) and Chromosome 21 Open Reading Matrix 29 (C2 lorf29), H olocarboxylase synthetase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; (c) determine the level of the genomic sequence of the fetus; and (d) compare the level of the fetal genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

Similarly, the method of the second aspect in this embodiment becomes: a method of detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) [omitted]; (b) treat DNA from a woman's biological sample, where the sample is bisulfite whole blood, serum, plasma, urine or saliva; (c) perform an amplification reaction using the DNA from step (b) and two primers to amplify a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit 2 (inhibitor) pseudogene 2 ( PPP1R2P2), Similarity to FemlA (Caenorhabditis elegans), CGI009, Carbonyl Reductase 1 (CBRI), Down Syndrome Cell Adhesion Molecule (DSCAM), Chromosome 21 Open Reading Matrix (C2 lorf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and wherein at least one of the two primers differentially binds to the fetal genomic sequence; and (d) comparing the level of the amplified portion of the genomic sequence from step (c) to a standard control, wherein an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

Similarly, the third aspect method in this embodiment becomes: a method for detecting and monitoring a disorder associated with pregnancy, comprising the steps of: (a) [omitted]; (b) treating DNA from a woman's biological sample, wherein the sample is whole blood, serum, plasma, urine or saliva, with a reagent that differentially modifies methylated and unmethylated DNAs; (c) determining the nucleotide sequence of a CpG-containing genomic sequence from step (b), wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGIl37, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit pseudogene 2 (inhibitor) (PPP1R2P2), similarity to FemlA {Caenorhabditis elegans), CGI009, carbonyl reductase 1 (CBRI), Down Syndrome cell adhesion molecule (DSCAM), chromosome 21 open reading matrix 29 (C21 or £ 29), Holocarboxylase Synthetase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) comparing the nucleotide sequence profile of step (c) with a standard control, wherein a change in the control pattern profile indicates the presence or progression of a disorder associated with pregnancy.

Similarly, the fourth aspect method in this embodiment becomes: a method for detecting trisomy 21 in a fetus in a pregnant woman, comprising the steps of: (a) [omitted]; (b) treating a woman's biological sample, wherein the sample is whole blood, serum, plasma, urine or saliva, with a reagent that differentially modifies methylated and unmethylated DNAs; (c) analyzing alleles of a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit pseudogene 2 (PPP1R2P2), FemlA (iCaenorhabditis elegans) similarity, CGI009, carbonyl reductase 1 (CBRI), Down Syndrome cell adhesion molecule (DSCAM), open reading matrix 29 of chromosome 21 (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132; and (1) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) determining the ratio of alleles, wherein a deviation from that of a woman carrying a fetus having no trisomy 21 indicates trisomy 21 in the fetus.

Similarly, the fifth aspect method in this embodiment becomes: a method of detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) [omitted]; (b) determine the level of a CpG-containing genomic sequence in a woman's biological sample, where the sample is whole blood, serum, plasma, urine or saliva, where the genomic sequence is at least 15 nucleotides in length, comprising at least least one unmethylated cytosine and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulator (inhibitor) subunit 2 pseudogene 2 (PPP1R2P2) and FemlA Similarity (Caenorhabditis elegans) and (2) a DNA sequence of no more than 10 kb upstream and / or downstream of the site; and (c) compare the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy.

Similarly, the sixth aspect method in the embodiment becomes: a method of detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) [omitted]; (b) determine the level of a CpG-containing genomic sequence in a woman's biological sample, where the sample is whole blood, serum, plasma, urine or saliva, where the genomic sequence is at least 15 nucleotides in length, comprising at least least one methylated cytosine and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI009, carbonyl reductase 1 (CBR1), Down Syndrome cell adhesion molecule ( DSCAM), open reading matrix 29 of chromosome 21 (C21orf29), Holocarboxylase Synthetase (HLCS) and CGI132 and (2) a DNA sequence of no more than 10 kb upstream and / or downstream of the site; and (c) compare the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy. BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Cloning and sequencing of CGI37 (A), region A and (B) bisulfite. B region between paired placental tissues and maternal blood cells.

Individual CpG sites are numbered across the first line, with defined nucleotide positions relative to chr21: 46.249.636 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser. Each subsequent line represents the state of methylation through the CpG sites in a cloning isolated single DNA molecule. Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively. Clones of placental tissue samples are labeled with a prefix "PLN," while those from maternal blood cells are labeled with a prefix "MBN." The placenta and maternal blood cells of the same pregnant person are identified by the identical sample number following "PLN" or "MBN."

Figure 2. Box plotting of methylation indices between all of the sequenced clones for placental and maternal blood cell samples for each of the CpG sites studied within CGI137 (A), region A and (B). region B. Through the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 46.249.636 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

Figure 3. Box plotting of methylation indices across all of the sequenced clones for placental and maternal blood cell samples for each of the CpG sites studied within PDE9A between third trimester and first trimester pregnancies for region A (A and B), region B (C and D) and region C (E). For Figures 3A and 3B, through the x-axis, the individual CpG sites are designated with their nucleotide positions relative to the chr21: 42.978.424 (+1) reverse filament of the Human May 2004 (hg17) assembly. UCSC Genome Browser; and for Figs. 3C and 3D, relative to chr21 direct filament: 42,978,718 (+1); for Fig. 3E, with respect to the direct filament of chr21: 42,978,005 (+1).

Figure 4. Box plotting of methylation indices between all of the sequenced clones for placental and maternal blood cell samples for each of the CpG sites studied within PPP1R2P2 region A between pregnancies (A), third trimester, and (B) . third quarter and (C). within region B of PPP1R2P2 between third trimester pregnancies. Across the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 36.180.493 (+1) from the May 2004 Human (hg17) assembly of the UCSC Genome Browser.

Figure 5. Box plotting of methylation indices between all of the sequenced clones for the placenta and maternal blood cell samples for each of the CpG sites studied within FemlA (C. elegans) Similarity (A), Region A, and ( B). region B. Through the x-axis, the individual CpG sites are designated with their nucleotide positions relative to chr21: 14,056,070 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

Figure 6. Box plotting of methylation indices among all of the sequenced clones for placental and maternal blood cell samples for each of the CpG sites studied within CGI009. Across the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 25,855,701 (+1) from the May 2004 Human (hg17) assembly of the UCSC Genome Browser.

Figure 7. Box plotting of methylation indices between all of the sequenced clones for the placenta and maternal blood cell samples for each of the CpG sites studied within Carbonyl reductase 1. Across the x-axis, individual CpG sites are designated. with their nucleotide positions relative to chr21: 36,363,538 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

Figure 8. Box plotting of methylation indices among all of the sequenced clones for the placenta and maternal blood cell samples for each of the CpG sites studied within Down syndrome cell adhesion molecule. Across the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 41,139,872 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

Figure 9. Box plotting of methylation indices between all of the sequenced clones for the placenta and maternal blood cell samples for each of the CpG sites studied within C21orf29. Across the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 44,953,288 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

Figure 10. Cloning and sequencing of CGIll bisulfite between paired placental tissues and maternal blood cells. Individual CpG sites are numbered across the first line, with defined nucleotide positions relative to chr21: 44,699,072 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser. Each subsequent line represents the state of methylation through the CpG sites in a cloning isolated single DNA molecule. Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively. Clones of placental tissue samples are labeled with a prefix "PLN," whereas maternal blood cells are labeled with a prefix "MBN." The placenta and maternal blood cells of the same pregnant person are identified by the identical sample number following "PLN" or "MBN."

Figure 11. Box plotting of methylation indices between all of the sequenced clones for placental and maternal blood cell samples for each of the CpG sites studied within CGI121. Across the x-axis, individual CpG sites are designated with their nucleotide positions relative to chr21: 45,262,112 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

Figure 12. Illustration of homogeneous MassEXTEND assay targeting the unmethylated form of CGI137. The nucleotide sequence spanning the amplified region is shown. The original DNA sequence is aligned above the converted disulphide sequence. CpG locations are identified by the "++" sign. The CpG sites are additionally numbered and the numbering corresponds to that in Fig. 1A and 2A and Table 2A. Cytosine residues that are not part of a CpG dinucleotide are identified by a ":" sign. The converted disulphide sequence represented is based on the assumption that all CpG sites are methylated. Alignments for forward, extension, and reverse primers are shown below the converted disulphide sequence.

Figure 13. Mass spectrometric plots of the homogeneous MassEXTEND assay targeting the unmethylated form of CGI137. Results for the mixture of pure placental DNA, maternal leukocyte layer DNA, 95: 5 (maternal leukocyte layer DNA: placental DNA), pre and postpartum maternal plasma and no pattern control (NTC) are shown. For all mass spectra, the χ axis represents the molecular weight of the detected extension products (shown as acute peaks), while the χ axis represents the intensity in arbitrary units. The expected position of the unmethylated molecule is as marked.

Figure 14. Combined Bisulfate Restriction Analysis (COBRA), analysis of (A). Holocarboxylase Synthetase Region A, (B). region B1 and (C). region B2. Two trisomy 21 placentae (T21 PLN), two normal placenta in the 10th trimester (Normal 1st PLN), two normal placentae in the 3rd trimester (Normal 3rd PLN) and two maternal blood cells in the 10th trimester (Leukocyte Layer) were analyzed. . PCR products were digested with (+) or without (-) BstU I enzyme. DNA methylation was detected by the appearance of the smaller digestion products. The increasing variation of one kb (Invitrogen Carlsbad, CA) (M) was used in gel electrophoresis.

Figure 15. COBRA analysis of CGI009. Two trisomy 21 placentae (T21 PLN), two normal 1st trimester placentae (Normal 1st PLN), two normal 3rd trimester placentae (Normal 3rd PLN), and two maternal blood cells in the 10th trimester (leukocyte layer) were analyzed. PCR products were digested with (+) or without (-) BstU I enzyme. DNA methylation was detected by the appearance of the smaller digestion products. The increasing variation of one kb (Invitrogen Carlsbad, CA) (M) was used in gel electrophoresis.

Figure 16. COBRA analysis of CGI132. two trisomy 21 placentae (T21 PLN), two normal placentae in the 1st trimester (Normal PLN1), two normal placentae 3rd trimester (Normal PLN3) and two maternal blood cells in the 10th trimester (leukocyte layer) were analyzed. PCR products were digested with (+) or without (-) BstU I enzyme. DNA methylation was detected by the appearance of the smaller digestion products. The increasing variation of one kb (Invitrogen Carlsbad, CA) (M) was used in gel electrophoresis.

Figure 17. HLCS region B2 bisulfite cloning and sequencing between placental tissues and maternal blood cells. Individual CpG sites are numbered across the first line, with defined nucleotide positions relative to the chr21-.37.274.682-37.275.036 reverse filament of the May 2004 Human (hgl7) assembly of the UCSC Genome Browser. Each subsequent line represents the state of methylation through the CpG sites in a cloning-isolated single DNA molecule. Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively. Trisomy 21 clones, normal and third trimester normal placental tissue samples are labeled with a prefix "PLN T21", "1st PLN Normal" and "3rd PLN Normal," respectively, while those from Maternal blood cells are labeled with a prefix "leukocyte layer." Placenta and maternal blood cells of different pregnant women are identified by sample numbers following the prefix.

Figure 18. Quantification of HLCS DNA from placental tissues and maternal leukocyte layer. Methylation index was defined as HLCS DNA concentrations after restriction enzyme over total concentrations as determined in the simulated digestion control of the same sample. Placental tissue DNA is labeled "Normal PLN," and that of the maternal leukocyte layer is labeled "MBC."

Figure 19. Detection of fetal specific HLCS in 3rd trimester maternal plasma. HLCS signals were detected in maternal plasma samples with (Figure 19A) or without (Figure 19B) methylation sensitive restriction enzyme treatment. Prepartum plasma samples are labeled "Pre," while postpartum plasma samples are labeled "Post." Restriction enzymes Hpa II and BstU I were used in digestion reactions and are labeled "(+)" in the plot. Simulated digestions without enzyme treatment are labeled "(-)."

DEFINITIONS

The term "pregnancy-associated disorder," as used in this application, refers to any condition or disease that may affect a pregnant woman, the fetus the woman is carrying, or both the woman and the fetus. Such a condition or disease may manifest its symptoms for a limited period of time, for example during pregnancy or childbirth, or it may last the entire lifetime of the fetus following its birthday. Some examples of a disorder associated with pregnancy include ectopic pregnancy, preeclampsia, premature birth and chromosomal abnormalities such as trisomy 21.

A "CpG-containing genomic sequence" as used herein refers to a DNA sequence segment at a defined location in an individual's genome, such as a human fetus or pregnant woman. Typically, a "CpG-containing genomic sequence" is at least 15 nucleotides in length and contains at least one cytosine. Preferably, it may be at least 30, 50, 80, 100, 150, 200, 250 or 300 nucleotides in length and contain at least 2, 5, 10, 15, 20, 25 or 30 cytosines. For any "CpG-containing genomic sequence" at a given location, for example within a centralizing region around a given genetic site on chromosome 21 (such as an island of CpG CGI137, PDE9A, CGI009, etc.), variations of nucleotide sequences may exist from individual to individual and from allele to allele even to the same individual. Typically, such a centralizing region around a defined genetic site (e.g., a CpG island) contains the site as well as upstream and / or downstream sequences. Each of the upstream or downstream sequences (5 'or 3' limit count of the genetic site, respectively) can be as long as 10 kb, in other cases it can be as long as 5 kb, 2 kb, 1 kb, 500 base pairs, 200 base pairs or 100 base pairs. In addition, a "CpG-containing genomic sequence" may encompass a transcribed or untranscribed nucleotide sequence for protein production, and the nucleotide sequence may be a protein coding sequence, a non-protein coding sequence (such as a protein promoter). transcription) or a combination thereof.

An "CpG island" in this application describes a DNA sequence segment found in a genome that has a minimum length, a minimum GC content, and a minimum observed CpG frequency / expected CpG frequency (OCF / ECF) ratio. . Yamada et al. (Genome Research 14: 247-266, 2004) described a number of standards for determining an island of CpG: these must be at least 400 nucleotides in length, have a GC content greater than 50% and an OCF / ratio. ECF greater than 0.6. Others (Takai et al., Proc. Natl. Acad. Sci. USA 99: 3740-3745, 2002) defined a CpG island less rigorously as a sequence of at least 200 nucleotides in length, having a GC content greater than 50% and an OCF / ECF ratio greater than 0.6. The concept of a "CpG island" on chromosome 21, as used in this application, is one that adjusts the CpG island profiles provided by any of the currently available computer programs designed for chromosome scanning based on the criteria set out above, covering the results obtained when using window size of 100, 200, or 300 nucleotides and displacement or step sizes of 1, 2, or 3 nucleotides in the evaluation process. The individual CpG islands mentioned in this disclosure are further defined by their corresponding genomic contig access number, GenBank version and region, the chromosomal site relative to Human May 2004 chromosome sequence (hgl7) assembly of the UCSC Genome Browser ( genome.ucsc.edu) and their ability to be amplified by PCR primers under given conditions, as indicated in Table 1 of this descriptive report.

The term "epigenetic state" or "epigenetic status" as used herein refers to any structural feature at a molecular level of a nucleic acid (eg, DNA or RNA) other than the primary nucleotide sequence. For example, the epigenetic state of a genomic DNA may include its secondary or tertiary structure determined or influenced, for example, by its methylation pattern or its association with cellular proteins.

The term "methylation profile" or "methylation status," when used in this application to describe the methylation status of a genomic sequence, refers to the characteristics of a DNA segment at a particular genomic site relevant for methylation. Such characteristics include, but are not limited to, if any of the cytosine (C) residues within this DNA sequence are methylated, location of C methylated residue (s), percentage of C methylated at any particular stretch of residues and allelic differences in methylation due to, for example, difference in the origin of alleles. The term "methylation profile" or "methylation state" also refers to the relative or absolute concentration of methylated C or unmethylated C at any particular stretch of waste in a biological sample.

The term "single nucleotide polymorphism" or "SNP" as used herein refers to the variation of polynucleotide sequence present in a single nucleotide residue within different alleles of the same genomic sequence. This variation may occur within the coding region or non-coding region (ie, promoter region) of a genomic sequence, if the genomic sequence is transcribed during protein production. Detecting one or more SNPs allows differentiation of different alleles from a simple genomic sequence.

The term "blood" as used herein refers to a blood sample or preparation of a pregnant woman or woman to be tested for possible pregnancy. The term encompasses whole blood or any blood fractions such as serum and plasma as conventionally defined. The term "bisulfite" as used herein encompasses all types of bisulfites, such as sodium bisulfite, which are capable of chemically converting a cytosine (C) to a uracil (U) without chemically modifying a methylated cytosine and thus may be used to differentially modify a DNA sequence based on the state of DNA methylation.

As used herein, a reagent that "differentially modifies" methylated or unmethylated DNA encompasses any reagent that modifies methylated and / or unmethylated DNA in a process whereby distinguishable products result from methylated and unmethylated DNA, thereby allowing identification of DNA methylation status. Such processes may include, but are not limited to, chemical reactions (such as a C -> U bisulfite conversion) and enzymatic treatment (such as cleavage by a methylation dependent endonuclease). Thus, an enzyme that preferably cleaves or digests methylated DNA is one capable of cleaving or digesting a DNA molecule at a much higher efficiency when DNA is methylated, whereas an enzyme that preferably cleaves or digests unmethylated DNA has an efficiency. significantly higher when DNA is not methylated.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in single or double stranded form. Unless specifically limited, the term embraces nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolised in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly embraces conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphism (SNPs), and complementary sequences as well as explicitly indicated sequence. Specifically, degenerate codon substitutions may be completed by generation of sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and / or deoxyoxy residues (Batzer et al., Nucleic Acid Res 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA and mRNA encoded by a gene.

The term "gene" means the segment of DNA involved in the production of a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) involved in gene product transcription / translation and transcription / translation regulation, as well as sequence intervention (introns) between individual coding segments (exons).

In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to an amino acid residue polymer. The terms apply to amino acid polymers wherein one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including life-size proteins (i.e. antigens), wherein amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are further modified, for example hydroxyproline, γ-carboxyglutamate and O-phosphoserine.

Amino acids can be referred to herein by the commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, too, may be referred to by their commonly accepted one-letter codes.

As used in this application, an "increase" or "decrease" refers to a detectable positive or negative change in the amount of an established standard control. An increase is a positive change, preferably at least 10%, more preferably 50%, even more preferably 2 times, even more preferably at least 5 times and more preferably at least 10 times the control value. Similarly, a decrease is a negative change, preferably at least 10%, more preferably 50%, even more preferably at least 80% and more preferably at least 90% of the control. Other terms indicating quantitative changes or differences on a comparative basis such as "plus" or "minus" are used in this application in the same manner as described above.

A "polynucleotide hybridization method" as used herein refers to a method for detecting the presence and / or amount of a polynucleotide based on its ability to form Watson-Crick base pairing under appropriate hybridization conditions with a polynucleotide probe of a known sequence. Examples of such hybridization methods include Southern blotting and Northern blotting.

"Primers" as used herein refer to oligonucleotides that may be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on the polynucleotide sequence that corresponds to a particular genomic sequence, for example, one located within the CpG island CGI137, PDE9A or CGI009 on chromosome 21, in various methylation states. At least one of the PCR primers for amplifying a polynucleotide sequence is sequence specific for the sequence.

"Standard control" as used herein refers to a sample comprising a genomic sequence of a predetermined amount or methylation profile (which may include multiple different and separable methylation related characteristics) suitable for use of a method of the present invention. , in order to compare the amount or state of methylation of a particular genomic sequence, for example, one located within the CpG island CGI137, PDE9A or CGI009 on chromosome 21, which is present in a test sample. A sample that serves as a standard control provides an average amount or methylation profile of a gene of interest that is typical for a definite time (eg, first trimester) during pregnancy in the blood of a normal, pregnant, healthy woman who carries a normal fetus, both of which are not at risk of developing any disorders or complications associated with pregnancy.

The term "normal," as used in the context describing a pregnant woman, refers to the fact that the woman is free from at least one condition of relevance, such as having a chromosomally abnormal fetus or suffering from an associated condition. with pregnancy (eg ectopic pregnancy, preeclampsia or premature birth). The term "normal," when used in another context, refers to certain characteristics, such as the methylation profile of a particular genomic sequence (for example, one located within the CpG island CGI137, PDE9A or CGI009 on chromosome 21) of both maternal and fetal origin found in a woman's blood, which is representative of a randomly selected group of healthy women who are pregnant with chromosomally normal fetuses and not susceptible to any pregnancy-related diseases or conditions. This selected group should comprise a sufficient number of women such that the average amount or methylation profile of the genomic sequence of interest among these women reflects, with reasonable accuracy, the corresponding profile in the general population of healthy pregnant women with healthy fetuses. In addition, the selected group of women generally have a gestational age similar to that of a woman whose blood is tested for an indication of a potential disorder associated with pregnancy. Preferred gestational age for practicing the present invention may vary depending on the disorder being evaluated. For example, a pregnant woman is assessed for the risk of preeclampsia preferably during the second trimester of pregnancy, whereas fetal chromosomal aneuploidy is preferably assessed and diagnosed as soon as possible. In addition, the preferred gestational age for the test may also depend on the gene of interest in the test.

The term "preeclampsia" as used herein refers to a condition that occurs during pregnancy, the main symptom of which consists of various forms of high blood pressure often accompanied by protein in the urine and edema (swelling). Preeclampsia, sometimes called pregnancy toxemia, is related to a more serious disorder called "eclampsia," which is preeclampsia along with seizures. These conditions usually develop during the second half of pregnancy (after 20 weeks), although they may develop shortly after birth or before 20 weeks of pregnancy.

The term "premature birth" or "premature birth" as used herein refers to the condition where labor that begins more than three weeks before the full gestation period of about 40 weeks, which often leads to premature birth. if not treated. The term "hyperemesis gravidarum" refers to extreme and persistent nausea and vomiting during pregnancy, particularly during the first trimester. Nausea and vomiting can lead to dehydration and prevent weight gain necessary for pregnancy.

An "ectopic pregnancy" refers to an abnormal pregnancy in which a fertilized egg has been implanted outside the womb. Although in most cases of ectopic pregnancy the egg settles in the fallopian tubes, this term also covers abnormal pregnancies where the fertilized egg is implanted in a woman's ovary, abdomen or cervix.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The presence of fetal DNA in maternal plasma was first reported in 1997 and offers non-invasive prenatal diagnosis simply by analyzing a maternal blood sample (Lo et al., Lancet 350: 485-487, 1997). So far, numerous potential clinical applications have been developed. In particular, quantitative abnormalities of maternal plasma fetal DNA concentrations have been observed to be associated with various disorders associated with pregnancy, including preeclampsia, preterm birth, pre-labor haemorrhage, invasive placentation, fetal Down syndrome and others. fetal chromosomal aneuploidies. Consequently, fetal DNA analysis in maternal plasma has been suggested as a potential marker for monitoring fetal-maternal well-being.

However, fetal DNA co-exists on the basis of maternal maternal plasma DNA. Consequently, more reported applications are relied upon to detect Y chromosome sequences as they are more conveniently distinguishable from maternal DNA. Such a method limits the applicability of existence assays to only 50% of each pregnancy, that is, those with male fetuses. Thus, there is a great need for the development of gender-independent fetal DNA markers for maternal plasma detection.

It has previously been shown that fetal and maternal DNA can be distinguished by their differences in methylation status (U.S. Patent Application Publication No. 20030044388). Methylation is an epigenetic phenomenon, which refers to processes that alter a phenotype without involving changes in the DNA sequence. By exploring the difference in DNA methylation status between the paternally and maternally inherited alleles in Hl 9, a site that presents a genomic impression (differential methylation and consequently differential expression of two alleles of a single gene, related to the paternal origin of a particular allele). ), one (YMD Lo) of the present inventors and their group first demonstrated the possibility of using epigenetic markers to detect maternally inherited DNA sequence derived from the maternal plasma fetus (Poon et al., Clin. Chem. 48: 35-41, 2002). Landes et al. also proposed the use of epigenetic markers for noninvasive prenatal diagnosis (U.S. Patent Application Publication No. 20030211522).

The present inventors have recently demonstrated that placental-derived RNA can be detected in maternal plasma (Ng et al., Proc. Natl. Acad. Sci. USA 100: 4748-4753, 2003). On the other hand, it has been shown that plasma DNA in normal individuals is predominantly derived from hematopoietic cells (Lui et al., Clin. Chem. 48: 421-427, 2002). Thus, it was hypothesized that the predominant source of maternal DNA is derived from peripheral blood cells while the placenta is a possible source of fetal DNA release in maternal plasma. Therefore, a strategy for the development of a generic fetal-specific DNA marker for detection in maternal plasma is to identify a gene that is differentially methylated between the placenta and the peripheral maternal blood cells. The present inventors demonstrated for the first time that various genomic sequences located at specific genomic sites on chromosome 21 are differentially methylated between fetal and fetal DNA (for example, the placenta) and maternal DNA of the mother's peripheral blood cells. Thus, this finding provides a new method for distinguishing fetal and maternal genomic DNA and new methods for noninvasive prenatal diagnosis.

II. General Methodology

The practice of this invention utilizes routine techniques in the field of molecular biology. Basic texts that disclose general methods of use in this invention include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in kilobases (kb) or base pairs (base pairs). These are estimated from acrylamide gel agarose or electrophoresis derivatives, sequenced nucleic acids or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

Oligonucleotides that are not commercially available may be chemically synthesized, for example according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981) using an automated synthesizer as described in Van Devanter et. al., Nucleics Acids Res. 12: 6159-6168 (1984). Oligonucleotide purification is performed using any technique recognized in the art, for example, natural acrylamide gel electrophoresis or anion exchanger high performance liquid chromatography (HPLC) as described in Pearson & Reanier, Chrotn. 255: 137-149 (1983).

The genomic sequences of the present invention, for example those located within the CpGs island on chromosome 21, such as CGI137, PDE9A and CGI009 and the synthetic oligonucleotide polynucleotide sequence can be verified using, for example, the termination method of chain for sequencing of double filament models by Wallace et al., Gene 16: 21-26 (1981).

III. Blood Sample Acquisition and DNA Extraction

The present invention relates to the analysis of the epigenetic state of fetal DNA found in maternal blood as a noninvasive means for detecting the presence and / or for monitoring the progress of a condition associated with pregnancy or disorder. Thus, the first steps of practicing this invention are to obtain a blood sample from a pregnant woman and extract DNA from the sample.

A. Acquisition of Blood Samples

A blood sample is obtained from a pregnant woman in a suitable pregnancy test period using a method of the present invention. The gestational period may vary depending on the disorder tested, as discussed below. A woman's blood draw is performed, usually following standard hospital or clinic protocol. An appropriate amount of peripheral blood, for example, typically between 5-50 ml, is collected and may be stored according to standard procedure even before preparation.

B. Preparation of Blood Samples

Analysis of fetal DNA found in maternal blood according to the present invention may be performed using, for example, whole blood, serum or plasma. Methods for preparing maternal blood serum or plasma are well known among those of skill in the art. For example, a pregnant woman's blood may be placed in a tube containing EDTA or a specialized commercial product such as Vacutainer SST (Becton Dickinson, Franklin Lakes, NJ) to prevent blood clotting and plasma can then be obtained from whole blood by centrifugation. On the other hand, serum may be obtained with or without centrifugation following blood coagulation. If centrifugation is used, then it is typically, though not exclusively, conducted at an appropriate speed, for example 1,500 - 3,000 χ g. Plasma or serum may be subjected to additional centrifugation steps before being transferred to a new tube for DNA extraction.

In addition to the acellular portion of whole blood, DNA can also be recovered from the cell fraction, enriched in the portion of the leukocyte layer, which can be obtained following centrifugation of a woman's whole blood sample and plasma removal.

C. DNA Extraction

There are numerous known methods for extracting DNA from a biological sample including blood. General DNA preparation methods (e.g., described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3a ed., 2001) may be followed; various commercially available reagents or kits such as QiaAmp DNA Mini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep ™ Blood DNA Isolation Kit (Promega, Madison, WI) and GFXᵀᴹ Genomic Blood DNA Purification Kit (Amersham , Piscataway, NJ) can also be used to obtain DNA from a blood sample from a pregnant woman. Combinations of more than one of these methods may also be used.

IV. Specific Chemical Modification of DNA Methylation

When extracted from a blood sample of a pregnant woman, DNA is treated with a reagent capable of chemically modifying DNA in a differential manner of methylation, ie different and distinguishable chemical structures will result from a cytosine residue (C). methylated residue and an unmethylated C residue following treatment. Such a reagent typically reacts with the unmethylated C residue (s) on a DNA molecule and converts each unmethylated C residue to a uracil (U) residue, whereas the methylated C residues remain unchanged. . This C —► U conversion enables detection and comparison of methylation status based on changes in the primary nucleic acid sequence. An exemplary reagent suitable for this purpose is bisulfite, such as sodium bisulfite. Methods for the use of bisulfite for chemical DNA modification are well known in the art (see, for example, Herman et al., Proc. Natl. Acad. Sci. USA 93: 9821-9826,1996) and will not be discussed in details here.

As a skilled person will recognize, any other reagents that are not named herein, but have the same property of chemically modifying methylated and unmethylated DNAs (or by any other mechanism) may be used to practice the present invention. For example, specific modification of DNA methylation may also be performed by methylation sensitive restriction enzymes, some of which typically cleave an unmethylated DNA fragment but not a methylated DNA fragment, while others (e.g., McrBC-dependent endonuclease methylation cleavage DNA containing methylated cytosines but not unmethylated DNA. In addition, a combination of chemical modification and restriction enzyme treatment, for example, combined bisulfite restriction analysis (COBRA), may be used to practice the present invention.

V. Amplification and Sequence Determination of

Polynucleotide

Following chemical modification of DNA in a different manner than methylation, the treated DNA is then subjected to sequence based analysis such that one or more of the genomic sequences of the present invention (for example, those located within the CpGs island on the chromosome) 21, such as CGI137, PDE9A and CGI009) of fetal DNA can be distinguished from their maternal DNA counterparts and that fetal genomic sequence methylation profile can be determined and compared to a standard control. In addition, since it is determined that a particular genomic sequence of fetal origin is hypermethylated or hypomethylated compared to the maternal counterpart, the amount of this fetal genomic sequence can be determined based on its specific methylation state. Subsequently, this amount can be compared to a standard control value and serves as an indication of the potential for a certain disorder associated with pregnancy.

A. Nucleotide Sequence Amplification

An amplification reaction is optional prior to sequence analysis for a genomic sequence after specific methylation modification. In some embodiments of this invention, amplification is performed to preferentially amplify a CpG-containing genomic sequence on chromosome 21 which has a particular methylation pattern, such that only the genomic sequence of a particular source, for example, a placenta or other fetal tissues are detected and analyzed.

A variety of polynucleotide amplification methods are well established and frequently used in research. For example, general polymerase chain reaction (PCR) methods for polynucleotide sequence amplification are well known in the art and thus are not described in detail herein. For a review of methods, protocols, PCR principles in primer design, see, for example, Innis, et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc. NY, 1990. Reagents and Protocols PCR are also available from commercial vendors such as Roche Molecular Systems.

PCR is most commonly performed as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is automatically cycled through a denaturation region, an annealing region and an extension reaction region. Machines specifically adapted for this purpose are commercially available.

Although PCR amplification of a target polynucleotide sequence (for example, a genomic sequence containing CpG on chromosome 21 where the fetal and maternal sequence is differentially methylated) is typically used in the practice of the present invention, one skilled in the art will recognize whereas amplification of a genomic sequence found in a maternal blood sample can be performed by any known method, such as ligase chain reaction (CSF), transcription-mediated amplification and self-sustaining sequence replication, or acid-based amplification. (NASBA), each of which provides sufficient amplification. The most recently developed branched DNA technology can also be used to qualitatively demonstrate the presence of a particular genomic sequence of this invention representing a particular methylation pattern or to quantitatively determine the amount of this particular genomic sequence in maternal blood. For a review of branched DNA signal amplification for direct quantification of nucleic acid sequences in clinical samples, see Night, Adv. Clin. Chem. 33: 201-235, 1998.

B. Polynucleotide Sequence Determination

Techniques for polynucleotide sequence determination are also well established and widely practiced in the relevant research field. For example, the basic principles and general techniques for polynucleotide sequencing are described in various research reports and treatises on recombinant molecular biology and genetics, such as Wallace et al, supra; Sambrook and Russell, supra and Ausubel et al., Supra. DNA sequencing methods routinely practiced in manual or automated research laboratories can be used to practice the present invention. Additional means suitable for detecting changes (e.g., C -> U) in a polynucleotide sequence for practicing the methods of the present invention include but are not limited to mass spectrometry, primer extension, polynucleotide hybridization, real time PCR. and electrophoresis.

SAW. Establishing a Standard Control

In order to establish a standard control for practicing the method of this invention, a group of healthy pregnant women carrying healthy fetuses is first selected. These women are of similar gestational period, which is within the appropriate timeframe of pregnancy to assess conditions such as preeclampsia, fetal chromosomal aneuploidy and preterm delivery using the methods of the present invention. Similarly, a standard control is established using samples from a group of healthy nonpregnant women.

The healthy state of selected pregnant women and the fetuses they are carrying is confirmed by well-established, routinely used methods that include, but are not limited to monitoring women's blood pressure, recording the onset of labor and conducting fetal genetic analysis using CVS and amniocentesis.

In addition, the selected group of healthy pregnant women carrying healthy fetuses should be of a reasonable size such that the average amount of a genomic sequence of this invention that originated from the fetus in the maternal blood or the methylation profile of the genomic sequence. The maternal blood fetal value obtained in the group can be reasonably considered to be representative of the normal or average amount or methylation profile among the general population of healthy women carrying healthy fetuses. Preferably, the selected group comprises at least 10 women.

A standard control for a fetal genomic sequence methylation profile may reflect different and separable multiple aspects of the methylation status of that particular genomic sequence. For example, one aspect of a methylation profile is any given methylated or unmethylated C residue; another aspect is the number of methylated C bases within a particular genomic sequence; An additional aspect of the profile represents the percentage (s) of methylated C at any given location. Additional aspects of a methylation profile may include, but are not limited to, the allelic difference in methylation, the ratio of differentially methylated and other alleles. The methylation profile of fetal genomic sequence may also vary depending on the type of tissue, for example placental or other fetal tissue. Thus, separate standard controls can be established for different fetal tissues used in the test.

Since an average level or methylation profile is established by a particular fetal genomic sequence present in the maternal blood based on the individual values found in each woman in the selected healthy control group, this average or representative value or profile or profile is considered to be one. standard control. Any blood sample containing a similar amount of the fetal genome sequence or a similar methylation profile of the fetal genome sequence can thus be used as a standard control. In addition, a solution containing a genomic DNA sequence in the average or representative average or quantity or the average or representative average or methylation profile can also be artificially assembled and serves as a standard control. In addition, separate standard controls may also be established by different aspects of the methylation profile of a genomic sequence of fetal origin.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be altered or modified to produce essentially the same or similar results.

Example 1

We aim to identify epigenetic markers that are fetal-specific in maternal blood. Previous data suggest that fetal DNA molecules in maternal plasma are predominantly placental derivatives (Chim et al., Proc Natl Acad Sci US A., 102: 14753-14758; Masuzaki et al., J. Med Genet 41, 289-292 , 2004; Flori et al., Hum Reprod 19, 723-724, 2004), whereas the DNA grounding in maternal plasma may originate from maternal blood cells (Lui et al., Clin Chem 48, 421-427, 2002). . Therefore, to identify fetal epigenetic markers, methylation profiles of genetic sites were evaluated in both placental and maternal blood cells with the intention of identifying sites that demonstrate differential methylation between the two tissue types. Such markers can be used for prenatal diagnosis and monitoring of pregnancy-related conditions.

MATERIALS AND METHODS

The identification of genomic sequence containing CpGs. DNA methylation refers to the addition of a methyl group to the fifth carbon position of cytosine residues in CpG dinucleotides. Clusters of such CpG dinucleotides on chromosome 21q were computationally identified through the UCSC Genome Browser Genome Bioinformatics Site (genome.ucsc.eduicgi-bin / hgGateway) (Yamada et al., Genome Res 14, 247-266, 2004). CpG sites were further unselected based on the criteria: a DNA sequence stretch of at least 400 bp in length with a minimum guanine and cytosine content of 50% and a minimum observed CpG frequency ratio / expected CpG frequency of at least 0.6. After CpG containing genomic sequences is identified, we also diversify to the CpG sites ahead and downstream to the previously identified genomic region, for example, PDE9A A and C regions.

Patient Recruitment and Sample Collection Placental tissues and corresponding blood samples were collected from women in the first and third trimesters of pregnancy. Placental tissues were collected from patients in the third trimester after cesarean delivery and from patients in the first trimester after termination of pregnancy. Maternal blood (10 ml) was collected in EDTA blood collection tubes prior to the commencement of labor or the performance of some obstetric procedures. Blood samples were centrifuged at 1600 χ g for 10 minutes at 4 ° C. The leukocyte layer portion was obtained after careful plasma removal and stored separately at -20 ° C. Placental tissues were rinsed in phosphate buffered saline and stored in flat polypropylene tubes at -80 ° C.

Bisulfite sequencing DNA was extracted from the placental tissues and maternal leukocyte layer using the QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), sample, 1 μg of DNA was subjected to bisulfite conversion, which converts unmethylated cytosine residues into uracil but leaves unchanged methylated cytosine residues by the CpGenoma DNA Modification Kit (Intergen, Burlington, MA) according to manufacturer's instructions. The bisulfite-converted DNA was then subjected to PCR amplification with primer pairs flanking the CpG sites (Table 1). Some CpG-containing genomic sequences are investigated by two or three PCRs, that is, regions A, B, and C. The primers were not designed not to be ligated to any potentially methylated cytosine residues. These primers are shown by way of illustration and should not be seen to limit the range of primers that may be used for this purpose. The reagents provided in the TaqMan PCR Core Reagent Kit (Applied Biosystems, Foster City, CA) were used. The reagent compositions for each PCR are detailed in Table 1. PCRs were typically performed in a final reaction volume of 25 μl with MgCl2, primers, TaqGold, Buffer IX, 200 μΜ of each dNTP, with or without dimethyl sulfoxide. (DMSO) or betaine. The thermal profile consisted of an initial denaturation step of 95 ° C for 10 minutes followed by 40 cycles of 95 ° C for 1 minute, an annealing temperature range of 55 to 65 ° C for 1 minute (Table 1). ° C for 1 minute and a final extension of 72 ° C for 10 minutes. To analyze the state of methylation in single molecule resolution, the PCR product was cloned into TA in a plasmid vector using the pGEM-T Easy Vector System (Promega, Madison, WI). Insertions of the positive recombinant clones were analyzed by cycle sequencing using the BigDye Terminator Cyele Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions. After purification by genCLEAN columns (Genetix), 8 μΐ of the samples were added to 12 μl Hi-Di formamide and proceeded on a 3100 DNA Analyzer (Applied Biosystems). Table 1. Identity, location, and primer sequences and PCR reaction conditions of the genomic sequences studied on chromosome 21. The respective regions in the genomic contigs (accession, version, and initial and final nucleotide numbers) deposited in GenBank of the National Center for Biotechnology Information and Chromosomal Locations (Chromosome, Starting and Ending Nucleotide Numbers) at the Human May 2004 (hgl7) assembly of the UCSC Genome Browser (available on the genome.ucsc.edu web site) are shown in the second and third columns respectively. <table> table see original document page 44 </column> </row> <table> <table> table see original document page 45 </column> </row> <table> Comparison of data and statistical analysis

A CpG site has been labeled methylated if the sequence is cytosine; marked as unmethylated if occupied by a thymine residue (uracil deoxide counterpart). The proportion of methylated cytosine residue for each CpG site was determined between placental tissues as well as maternal blood samples. The distribution of methylated and unmethylated cytosine was compared between placental tissues and maternal leukocyte layer for each CpG site by chi-square analysis. The P value of <0.05 is considered statistically and significantly different. Statistical analysis was performed using Sigma Stat 3.0 software (SPSS).

RESULTS AND DISCUSSION

Among the genomic sequences containing CpGs identified from the computational research, 13 sites were the focus of the present investigation. The names, chromosomal site, and GenBank Accession Numbers of these sites are listed in Table 1. For each of the sites studied, bisulfite sequencing was performed on the placental tissues and maternal blood cells.

The methylation profile of CGI137 was studied between placental tissues and corresponding maternal blood cells collected from 5 third trimester pregnancies. Bisulfite sequencing data for regions A and B of all 5 cases are shown in Figures 1A and 1B, respectively. Each row in the respective panels represents the result of bisulfite sequencing for a clone while each column represents an individual CpG dinucleotide within the genomic sequence. The proportion of methylated clones among all those of the sequenced clones, the methylation index at each CpG site was determined for all 5 placental tissue and maternal blood cell samples. Data are summarized in Figures 2A and 2B. It can be seen that the placenta is hypomethylated compared to maternal blood cells. Chi-square analysis was performed to compare the distribution of methylated and unmethylated clones between placental tissues and maternal blood cells at each CpG site. Differences in methylation indices between placenta and maternal blood cells are statistically significant for all 21 CpG sites (Chi-square, P <0.0001, Tables 2A and 2B).

Table 2A. Summary of bisulfite sequencing data and the chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells at individual CpG sites within Region A CGI137. Individual CpG sites are designated by their nucleotide positions relative to chr21: 46.249.636 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 47 </column> </row> <table> Table 2B. Summary of bisulfite sequencing data and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells at individual CpG sites within Region B of CGI137. Individual CpG sites are designated by their nucleotide positions relative to chr21: 46.249.636 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 48 </column> </row> <table>

Phosphodiesterase 9A (PDE9A) The methylation profile of PDE9A was studied between placental tissues and the corresponding maternal blood cells collected from third trimester pregnancies and 5 first trimester pregnancies. Bisulfite cloning and sequencing were performed and the methylation indexes of CpG sites studied in placental tissues and maternal blood cells for amplicon region A are summarized in Figures 3A and 3B. Corresponding data for region B is summarized in Figures 3C and 3D. Comparisons between third trimester placental tissues and maternal blood cells for region C are shown in Figure 3E. In general, the placenta is hypomethylated compared to maternal blood cells. Chi-square analysis was performed as described above to statistically evaluate significant differences between the two tissues. Approximately CpG2250 in the third trimester comparison is statistically significantly different in both first trimester and third trimester pregnancies (chi-square analysis, Tables 3A to E). Table 3A. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of third trimester placental tissues and maternal blood cells at individual CpG sites within PDE9A region A. Individual CpG sites are designated by their nucleotide positions relative to the chr21 reverse filament: 42,978,424 (+1) from Human May 2004 (hgl7) UCSC Genome Browser assembly

<table> table see original document page 49 </column> </row> <table>

Table 3B. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells in the first trimester at individual CpG sites within PDE9A region A. Individual CpG sites are designated by their nucleotide positions relative to the chr21 reverse strand: 42,978,424 (+1) of the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 49 </column> </row> <table> Table 3C. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and third trimester maternal blood cells at individual CpG sites within PDE9A region B. Individual CpG sites are designated by their nucleotide positions relative to chr21: 42,978,718 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser. <table> table see original document page 51 </column> </row> <table>

<table> table see original document page 51 </column> </row> <table> 3D table. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells in the first trimester at individual CpG sites within PDE9A region B. Individual CpG sites are designated by their nucleotide positions relative to chr21: 42,978,718 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 52 </column> </row> <table> Table 3E. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and third trimester maternal blood cells at individual CpG sites within the PDE9A C region. Individual CpG sites are designated by their nucleotide positions relative to chr21: 42,978,005 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 53 </column> </row> <table>

Homo sapiens 1 protein phosphatase, pseudogene 2 of regulatory (inhibitory) subunit 2 (PPP1R2P2). The methylation profile of PPP1R2P2 region A was studied between placental tissues and corresponding maternal blood cells collected from 5 third trimester pregnancies and 5 first trimester pregnancies. Bisulfite cloning and sequencing were performed and the methylation indices of the CpG sites studied for region A in placental tissues and maternal blood cells are summarized in Figures 4A and 4B. In general, the placenta is hypomethylated compared to maternal blood cells. Chi-square analysis was performed as described above to statistically evaluate significant differences between the two tissues. Among the CpG sites studied, 23 are statistically and significantly different in third trimester pregnancies and 23 are statistically and significantly different in first trimester pregnancies (Chi-square analysis, Tables 4A and 4B). In addition, the methylation profile of CpG sites in PPP1R2P2 B region was also analyzed by cloning and sequencing of bisulfite in placental tissues and the corresponding maternal blood cells collected from 5 third trimester pregnancies. For most CpG sites in region B, placental tissues are hypomethylated compared to maternal blood cells. The methylation indices in region B are summarized in Figures 4C. Chi-square analysis was performed as described above to statistically evaluate significant differences between the two tissues. Among the 12 CpG sites studied, 5 are statistically significantly different (Chi-square analysis, Tables 4C).

Table 4A. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and third trimester maternal blood cells at individual CpG sites within PPP1R2P2 region A. The individual CpG sites are designated by their nucleotide positions relative to chr21: 36,180,493 (+1) from the May 2004 Human (hg17) assembly of the UCSC Genome Browser. <table> table see original document page 55 </column> </row> <table> <table> table see original document page 56 </column> </row> <table>

Table 4B. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells in the first trimester at individual CpG sites within PPP1R2P2 region A. The individual CpG sites are designated by their nucleotide positions relative to chr21: 36,180,493 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser. <table> table see original document page 57 </column> </row> <table>

<table> table see original document page 57 </column> </row> <table> Table 4C. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells in the first trimester at individual CpG sites within PPP1R2P2 region B. The individual CpG sites are designated by their nucleotide positions relative to chr21: 36,180,493 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 58 </column> </row> <table>

FemlA Similarity (<Caenorhabditis elegans (C. elegans)) The FemlA (C. elegans) Similarity methylation profile was studied between placental tissues and the corresponding maternal blood cells collected from the 5 third trimester pregnancies. Bisulfite cloning and sequencing were performed and the methylation indices of CpG sites studied in placental tissues and maternal blood cells are summarized in Figures 5A and 5B for regions A and B, respectively. In general, the placenta is hypomethylated compared to maternal blood cells. Chi-square analysis was performed as described above to statistically evaluate significant differences between the two tissues. Among the CpG sites studied, 8 are statistically and significantly different in region A and 7 are statistically and significantly different in region B (Chi-square analysis, Tables 5A and 5B). Table 5A. Summary of bisulphite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of third trimester placental tissues and maternal blood cells at individual CpG sites within Fem1A-like region (C. elegans) . Individual CpG sites are designated by their nucleotide positions relative to chr21: 14,056,070 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 59 </column> </row> <table>

Table 5B. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and third trimester maternal blood cells at individual CpG sites within Fem1A-like B region (c. Elegans) . Individual CpG sites are designated by their nucleotide positions relative to chr21: 14,056,070 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 59 </column> </row> <table>

CGI009 The methylation profiles of CGI009 from 2 maternal blood cell samples were compared with those from 2 first and third trimester placental tissue samples collected from normal pregnancies as well as 2 first trimester placental tissue samples from pregnancies. fetus has trisomy 21. Bisulfite cloning and sequencing were performed and the methylation indices of CpG sites studied in placental tissues and maternal blood cells are summarized in Figure 6. Overall, the placenta is hypermethylated when compared to maternal blood cells .

Carbonyl Reductase I (CBR1) The CBRl profiles of 2 maternal blood cell samples were compared with those of 2 first trimester placental tissue samples collected from normal pregnancies as well as 2 first trimester placental tissue samples from pregnancies. fetus had trisomy 21. Bisulfite cloning and sequencing were performed and the methylation indices of CpG sites studied in placental tissues and maternal blood cells are summarized in Figure 7. In general, the placenta is hypermethylated when compared to blood cells maternal Among the 20 CpG sites studied, all are statistically and significantly different between maternal blood cells and first trimester placental tissues of both normal and trisomy 21 (Chi-square analysis, Table 6).

Table 6. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of normal and trisomy 21 first trimester maternal blood cells at individual CpG sites within CBR1. The individual CpG sites are designated by their nucleotide positions relative to chr21: 36,363,538 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 60 </column> </row> <table> Down Syndrome Cell Adhesion Molecule (DSCAM) DSCAM methylation profiles of 2 maternal blood cell samples were compared with those from 2 first trimester and 2 third trimester placental tissue samples collected from normal pregnancies as well as 2 first trimester placental tissue samples from pregnancies where the fetus had trisomy 21. Bisulfite cloning and sequencing were performed and The methylation of the studied CpG sites in the placental tissues and maternal blood cells are summarized in Figure 8. In general, the placenta is hypermethylated compared to maternal blood cells. Among the 27 CpG sites studied, all are statistically and significantly different between maternal blood cells and first trimester placental tissues from both normal and trisomy 21 pregnancies (Chi-square analysis, Table 7).

Table 7. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profiles of normal and trisomy 21 first trimester maternal blood cells at individual CpG sites within DSCAM. Individual CpG sites are designated by their nucleotide positions relative to chr21: 41,139,872 (+1) from the Human of May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 61 </column> </row> <table> Chromosome 21 open reading frame 29 (C21orf29) The C21orf29 methylation profiles of 2 maternal blood cell samples were compared with that of 1 first trimester placental tissue sample and 2 third trimester samples collected from normal pregnancies as well as 2 first trimester placental tissue samples from pregnancies where the fetus had trisomy 21. Bisulfite cloning and sequencing were performed and The methylation of the studied CpG sites in the placental tissues and maternal blood cells are summarized in Figure 9. In general, the placenta is hypermethylated when compared to maternal blood cells. Among the 16 CpG sites studied, all 9 and 10 sites are statistically and significantly different between maternal blood cells and first trimester placental tissues of trisomy 21 pregnancies, first trimester and third trimester placental tissues of normal pregnancies, respectively. (Chi-square analysis, Table 8).

Table 8. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of normal and trisomy 21 first trimester maternal blood cells at individual CpG sites within C21or / 29 . Individual CpG sites are designated by their nucleotide positions relative to chr21: 44,953,288 (+1) from the Human May 2004 (hgl7) assembly of the UCSC Genome Browser.

<table> table see original document page 62 </column> </row> <table> Genetic sites that showed no differential methylation pattern between placental tissues and maternal blood cells Differential methylation between placental tissues and maternal blood cells was universally observed for all studied sites. The methylation profile of CGIlll was studied between placental tissues and corresponding maternal blood cells collected from 4 third trimester pregnancies. Cloning and sequencing of bisulfite were performed and the results are shown in Figure 10. Clones of both maternal blood cells and placental tissues were predominantly unmethylated.

Similarly, the methylation profile between maternal blood cells and placental tissues of 5 third trimester pregnancies were not significantly different for CGI21 except at the CpG 1603 site (Figure 11 and Table 9).

Table 9. Summary of bisulfite sequencing data, methylation indices, and chi-square analysis comparing the methylation profile of placental tissues and maternal blood cells in the first trimester at individual CpG sites within CGI21. The individual CpG sites are designated by their nucleotide positions relative to chr21: 45,262,112 (+1) from the May 2004 Human (hgl7) assembly of the UCSC Genome Browser. <table> table see original document page 64 </column> </row> <table> On the other hand, for KIAA0656, protein-binding factor 2 (HSF2BP) and COL6A1 heat shock transcription, placental tissues and blood cells Both maternal mothers studied were predominantly methylated with no significant difference between the methylation index.

Example 2

Genomic sequences containing a differential methylation profile between placental tissues and maternal blood cells are useful as fetal epigenetic markers for detection of maternal blood. In order to distinguish fetal-derived DNA molecules from those that are maternally derived in maternal blood, assays should target the detection of the placental-specific epigenetic form of each differentially methylated site. An assay was developed to target the placenta specific form of CGI137 (Figure 1), that is, unmethylated molecules and assessed their specificity.

MATERIALS AND METHODS

Sample processing and DNA extraction. A third trimester placental tissue sample and corresponding maternal blood sample were collected from a patient undergoing delivery by elective cesarean section. Maternal blood (10 ml) was collected in EDTA tubes before as well as after delivery. Blood samples were centrifuged at 1600 x g for 10 minutes at 4 ° C. The leukocyte layer portion was obtained after careful plasma removal and stored separately at -20 ° C. Plasma was further centrifuged at 16,000 x g for 10 minutes and 1.6 ml of plasma was transferred to clean polypropylene tubes being careful not to disturb the underlying cell granule. Placental tissue biopsies were rinsed in phosphate buffered saline and stored in flat polypropylene tubes at -80 ° C. DNA was extracted from placental tissues using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Maternal plasma and leukocyte layer DNA were extracted by the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For each placental and leukocyte layer DNA sample, 1 μg of DNA was subjected to bisulfite conversion, which converts unmethylated cytosine residues into uracil, but leaves the methylated cytosine residues unchanged by the Methilampju DNA Modification Kit (Epigentek). Inc., New York, NY) according to the manufacturer's instructions. DNA extracted from 1.6 ml of plasma was also subjected to bisulfite conversion. Additional aliquots of mixed bisulfite converted DNA were also prepared comprised of mixtures of the leukocyte layer and Placental DNA Preparations in the ratio of 95: 5.

Homogeneous MassEXTEND Assay Just as placental tissues are hypomethylated relative to maternal blood cells in CGI137 (Figures 1 and 2), an assay targeting detection of the unmethylated form of CGI137 has been designed, the assay is based on the MassARRAY platform (Sequenom , San Diego, CA). Specific methylation primers are designed for specific amplification of the unmethylated form of CGI137. An illustration of the primer location is shown in Figure 12 and the primer sequences are listed in Table 10. The forward primer transposes the CpG sites 472, 477, 481, and 486. The reverse primer transposes the CpG sites 553 and 561. The amplicon transposes between the coordinates: 2742998-2743130 for GenBank Access: NT_ 011515. A primer extension assay (Figure 12) based on the homogeneous MassEXTEND (hME) protocol is designed to interrogate the methylation status of the CpG 541 site. Basically, the primer extension primer transposes to nucleotide 3 "to the CpG 541 site (Table 10). An unmethylated molecule should be extended by a nucleotide with the incorporation of a dd (2 ', 3'-dideoxynucleoside) ATP, while a methylated molecule must be extended by two nucleotides with the incorporation of a dGTP followed by a ddATP. The unmethylated and methylated molecules are subsequently detected and resolved by their differences. mass differences in a MassARRAY Analyzer Compact (Sequenom) mass spectrometry.

Table 10. The primer sequences of the hME assay targeting the unmethylated form of CGl 137.

<table> table see original document page 67 </column> </row> <table>

Three μl of bisulfite converted DNA from placental samples (equivalent to 150 ng DNA prior to bisulfite conversion), and maternal leukocyte layer and mixtures as well as ten μl. of bisulfite - converted DNA plasma (DNA equivalent extracted from 1.6 ml maternal plasma) were amplified for each 25 μl PCR reaction. Each reaction contained PCR IX buffer (Applied Biosystems, Foster City, CA) with 2.0 mM MgCl2 (Applied Biosystems), 200 μΜ each of dATP, dGTP and dCTP, 400 μM dUTP (Applied Biosystems), 200 nM each of forward and reverse primers (Integrated DNA Technologies, Coralville, IA) and 1 U of AmpliTaq Gold® DNA Polymerase (Applied Biosystems). The PCR reaction was initiated at 95 ° C for 10 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 40 seconds, extension at 72 ° C for 40 seconds for 40 cycles and a final incubation at 72 ° C for 5 minutes.

The PCR products were subjected to a shrimp alkaline phosphatase treatment to dephosphorylate any remaining dNTPs and prevent their incorporation into the subsequent primer assay extension. For each 25 μL PCR reaction, 0.34 pL hME buffer (Sequenom), 0.6 μL shrimp alkaline phosphatase (Sequenom) and 3.06 μL water were added. The reaction mixture was incubated at 37 ° C for 40 minutes, followed by heat inactivation at 85 ° C for 5 minutes.

For the primer extension reaction, 4 μL of base extension reaction cocktail containing 1500 nM extension primer (Integrated DNA Technologies), 1.15 U of Thermossequenase (Sequenom) and 64 μΜ each of ddATP, ddCTP , dTTP and dGTP (Sequenom) were added to 10 μl of the PCR products. The reaction condition was 94 ° C for 2 minutes, followed by 94 ° C for 5 seconds, 52 ° C for 5 seconds and 72 ° C for 5 seconds for 75 cycles.

The final base extension product was purified with SpectroCLEAN resin (Sequenom) to remove salts that may interfere with mass spectrometry analysis. Twenty-four microliters of water and 12 mg of resin were added in each base extension product. The final mixtures were mixed on a rotator for 20 minutes. After centrifugation at 361 g for 5 minutes, approximately 10 nL of reaction solution was dispensed into a SpectroCHIP format 384 (Sequenom) pre-labeled with a 3-hydroxypicolinic acid matrix using a SpectroPoint nanodispenser (Sequenom). A MassARRA Y® Compact Mass Spectrometer Analyzer (Sequenom) was used for the acquisition of SpectroCHIP data. Mass spectrometric data was automatically imported into SpectroTYPER (Sequenom) base data for analysis.

RESULTS AND CONCLUSION

Mass spectra for MassARRAY analyzes are shown in Figure 13. Primer extension product for the unmethylated form of CGI137 can be detected in placental and prepartum DNA plasma samples as well as 95: 5 layer mixtures leukocyte disease and placental DNA. These data suggest that the assay is sensitive to detecting the unmethylated placenta derived form of CGI137 in maternal plasma and DNA mixtures up to a fractional concentration of 5%. No signs were detected postpartum of the maternal plasma sample confirming the specificity of pregnancy of the unmethylated form of CGI137. The lack of signal in the maternal leukocyte layer sample also confirms the specificity of the assay towards detecting the unmethylated form of CGI137. In summary, due to differences in the methylation profile of CGI13 between placental tissues and maternal blood cells, sensitive and specific assays can be developed to target detection of the placental derivative form of CGI137 among a background of blood cell derived methylated CGI13 molecules. maternal Since the placenta is a source of fetal DNA release tissue in the maternal circulation (Chim et al., Supra), where the latter contains a superior foundation of maternal blood cell DNA (Liu et al., Supra), Assays targeting placental specific epigenetic markers are useful for the detection of fetal specific nucleic acid molecules in maternal blood.

Example 3

An alternative method, combined bisulfite restriction analysis (COBRA) (Xiong and Laird Nucleics Acids Res 25: 2532-2534, 1997), was used to evaluate the presence of differential methylation of 3 genomic sequences on chromosome 21 (Table 11) between Placental DNA and maternal blood cells.

Table 11. Identity, location, primer sequences, and PCR reaction conditions of the genomic sequences studied on chromosome 21. The respective regions in the genomic contigs (accession number, version number, beginning and end nucleotide) deposited in the GenBank of the National Center for Biotechnology Information and chromosome locations (chromosome numbers, nucleotide start and end) in the Human May 2004 (hgl7) assembly of the UCSC Genome Browser (genome.ucsc.edu) are shown in the second and third columns, respectively. <table> table see original document page 70 </column> </row> <table> MATERIALS AND METHODS

Combined bisulfite restriction analysis (COBRA). One μg of DNA was subjected to a bisulfite conversion by the EZ DNA Methylation Kit (Zymo Research, Orange, CA) according to the manufacturer's instructions. Forty nanograms of bisulfite converted DNA (based on the input of unconverted DNA) was then subjected to PCR amplification as described in Example 1, with some modifications. The reagents provided in the HotStar Taq DNA Polymerase Kit (Qiagen, Hilden, Germany) were used. Reagent compositions for each PCR are detailed in Table 11. PCR was typically performed in a final reaction volume of 20 μl with MgCl2, primers, HotStar Taq, PCR Buffer IX, 50 μΜ of each dNTP and 2X PCRx Enhancer. (Invitrogen, Carlsbad, CA). The thermal profile consisted of an initial denaturation step of 95 ° C for 15 minutes, followed by 50-55 cycles of 95 ° C for 20 seconds, 58 or 60 ° C for 30 seconds (Table 11), 72 ° C for 1 , 5 minutes and a final extension of 72 ° C for 3 minutes. The PCR products were then subjected to restriction enzyme digestion. The restriction enzyme to be used for each respective site was chosen for its ability to distinguish between methylated and unmethylated sequence after bisulfite conversion. In essence, restriction sites are present only in the methylated or unmethylated sequence, but not both, so that one sequence must be digested while the other should remain intact (Table 12). Restriction enzyme digestions were performed in a final reaction volume of 20 μl with 5 μl of PCR products, appropriate IX buffer and 10U restriction enzyme (or none for simulated digestion) at manufacturers recommended temperatures for 2 hours. All enzymes were purchased from New England Biolabs (Beverly, MA). The digested products were then analyzed by gel electrophoresis. Enzyme Cut state_methylacetate Complete digestion products (base pairs)

Table 12. Result Prediction for COBRA Analysis

<table> table see original document page 72 </column> </row> <table>

Cloning and sequencing of bisulfite. DNA from the same PCR amplification reaction as described in the "Combined Bisulfite Restriction Analysis (COBRA)" session shown above was used for bisulfite cloning and sequencing. To analyze the methylation state at single molecule resolution, the PCR product was cloned into TA in a plasmid vector using the pGEM-T Easy Vector System (Promega, Madison, WI). Insertions of positive recombinant clones were analyzed by cycle sequencing using the BigDye Terminator Cycle Sequencing ν1.1 kit (Applied Biosystems) according to the manufacturer's instructions. After purification by ethanol precipitation, samples were resuspended by 10 µl Hi-Di formamide and conducted on a 3100 DNA Analyzer (Applied Biosystems).

RESULTS AND CONCLUSION

Holocarboxylase Synthetase (HLCS). The HLCS putative promoter region methylation profiles of 2 maternal blood cell samples were compared with those of 2 first and third trimester placental tissue samples collected from normal pregnancies as well as 2 first trimester placental tissue samples. of pregnancies with trisomy 21. The COBRA assay was performed and data for gel electrophoresis for region A, region B, and region B2 are shown in Figures 14A, 14B, and 14C, respectively. In general, the placenta is hypermethylated compared to maternal blood cells. The bisulfite cloning and sequencing experiment was performed in the B2 region to further analyze the methylation state in single molecule resolution and the result shown in Figure 17 confirmed that HLCS methylation is placental specific.

CGI009. The CGI009 methylation profiles of 2 maternal blood cell samples were compared with those of 2 first and third trimester placental tissue samples collected from normal pregnancies as well as 2 first trimester placental tissue samples from trisomy pregnancies. 21. The COBRA assay was performed and gel electrophoresis data are shown in Figure 15. In general, the placenta is hypermethylated compared to maternal blood cells.

CGI132. The CGI132 methylation profiles of 2 maternal blood cell samples were compared with those of 2 first and third trimester placental tissue samples collected from normal pregnancies, as well as 2 first trimester placental tissue samples from trisomy pregnancies. 21. The COBRA assay was performed and gel electrophoresis data are shown in Figure 16. In general, the placenta is hypermethylated compared to maternal blood cells.

Example 4

Based on the differential methylation characteristic of the markers identified in the placental tissues and maternal blood cells, an alternative method using methylation-sensitive restriction enzyme digestion followed by real-time quantitative PCR was developed to quantitatively analyze the differential methylation of the genomic sequence. HLCS region B2 (Table 11) between placental DNA and maternal blood cells.

MATERIALS AND METHODS

Methylation-sensitive restriction enzyme digestion. DNA was extracted from placental tissues using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Maternal leukocyte layer DNA and plasma were extracted by the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For each placental and leukocyte layer DNA sample, 100 ng DNA was subjected to methylation-sensitive restriction enzyme digestion. Restriction enzyme digestions were performed in a final reaction volume of 50 μl, with DNA, appropriate buffer IX and 25U of Hpa II and 5OU of BstU I (or none for simulated digestion) at temperatures recommended by manufacturers for at least 16 hours For each maternal plasma sample, 1.6 ml of plasma was used for DNA extraction and was eluted in 50 μΐ of deionized water, 21 μΐ of which were subjected to restriction enzyme digestion. Enzyme digestions were performed in a final reaction volume of 30 μΐ, with DNA, appropriate Hpa II and 3 OR BstU I buffer (IX and 20U) (or none for simulated digestion) at temperatures recommended by manufacturers for at least 16 hours All enzymes were purchased from New England Biolabs (Beverly, MA). The digested products were then analyzed by quantitative real time PCR. Selected restriction enzymes digest only unmethylated DNA, but not methylated DNA. Since the data from Example 3 has shown that HLCS is hypermethylated in placental tissues and hypomethylated in maternal blood cells, we expect a proportion of placental tissue DNA to remain detectable while more maternal blood cell DNA must be digested and thus , undetectable after restriction enzyme treatment.

Real-time quantitative PCR. The real time PCR assay was developed for quantitative analysis of HLCS genomic DNA with and without restriction enzyme digestion. 4 μl of restriction enzyme treated DNA or simulated digestion sample was used in the real time PCR assay. Each reaction contains IX TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 300 nM forward primer (5'-CCGTGTGGCCAGAGGTG-3 '), 300 nM reverse primer (5'-TGGGAGCCGGAACCTACC-3') and 100 nM nM TaqMan probe (5'-6FAM-TCCCGACCTGGCCCTTTGCC-TAMRA-3 '). The thermal profile was 50 ° C for 2 minutes, 95 ° C for 10 minutes, 50 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute. All reactions were conducted in duplicate and the average amount was taken. Serially diluted human genomic DNA originally quantified by optical density measurement was used as the quantitative standard for the assay. Just as the HLCS DNA detectable after restriction enzyme digestion represented the methylated fraction, we expressed quantitative real-time PCR as a methylation index. The methylation index of a sample is calculated by dividing the DNA HLCS concentration after enzyme digestion with that obtained with simulated digestion.

RESULTS AND CONCLUSION

The putative HLCS promoter region methylation profiles of the 8 maternal blood cell samples were compared with those of 2 first trimester and second trimester placental tissue samples collected from normal pregnancies. Restriction enzyme digestion followed by real-time PCR analysis was performed and the results are shown in Figure 18. DNA from all maternal blood cell samples were mostly digested by restriction enzymes, resulting in indices of methylation approaching 0; whereas placental tissues were partially digested, resulting in methylation indices ranging from 0.567 to 0.966.

Previous data suggest that placenta is the predominant source of fetal DNA tissue while maternal blood cells represent the major contributor to the maternal DNA background that is detectable in maternal plasma. We believe that the placenta-specific fraction (with reference to maternal blood cells) of HLCS DNA, that is, the methylated or non-digestible fraction can be detected in maternal plasma and is pregnancy-specific. Paired pre- and postpartum 3rd trimester plasma samples from 25 normal pregnant people were recruited. Restriction enzyme digestion followed by real time PCR assay was performed and the results are shown in Figure 19. The HLCS signal was shown to be positively detected in the 3rd trimester plasma samples of the digested prepartum enzyme. The average HLCS concentration in postpartum plasma samples after enzyme digestion was 8.1% of that HLCS concentration medium in the postpartum plasma samples after enzyme digestion (Figure 19A). Standard clearance of the enzyme treated pre- and postpartum plasma indicates that the detected HLCS signal is specific. In contrast, the mean HLCS DNA concentration in postpartum maternal plasma with simulated restriction enzyme digestion was 83.8% of that in simulated digested prenatal samples (Figure 19B).

All patents, patent applications, and other publications cited in this application, including published amino acid or polynucleotide sequences, are incorporated by reference in their entirety for all purposes. SEQUENCE LIST

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<213> Artificial Sequence <220>

<223> F- region A primer CGJ137 <40Q> 6

ggttgggttg aggagggta gt 22

<210> 7 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> R- region A primer CGJ137

<400> 7

accocraacc ertctctaee tacaa

25 <210> 8 <211> 29 <212> DNA

<213> Artificial Sequence

<220>

<223> Region B F-primer CG137

<400> 8

aaggggagtt gagatattgt agggtttat 29

<210> 9 <211> 23 <212> DNA

<213> Artificial Sequence

<220>

<223> B-region R-primer CG137

<210> 10 <211> 26 <212> DNA

<213> Artificial Sequence

<220>

<223> F-primer of phosphodiesterase 9A region A (PDE9A)

<400> 10

gtttttaggg agggggtatt tygagt 26

<210> 11 <211> 36 <212> DNA

<213> Artificial Sequence

<220>

<223> P-phosphodiesterase A-region R-primer (PDE9A)

<400> 9

aacacctaaa aaactcrccr aaa

23

<400> 11

aatctatttt ctatatttca ctatttccaa ataaaa

36

<210> 12 <211> 36 <212> DNA

<213> Artificial Sequence

<220>

<223> P-phosphodiesterase B region F-primer (PDE9A)

<400> 12

gtatgtatta attaaatgaa aagatgagtt tgtgat

36

<210> 13 <211> 25 <212> DNA

<213> Artificial Sequence <220>

<223> R-primer of foEfodiesterase 9A region (PDS9A)

<400> 13

creaaaacce cttataaaaa. goshawk 25

<210> 14

<211> 27

<212> one

<213> Artificial Sequence

<220>

<223> F-C region phosphodiesterase 9A primer {PDE9 A)

<400> 11

ggtggttgtg tgtgttt ^ gt ttttagt 27

<21Ó> 15

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> R-primer gives phosphodiesterase 9A region C (FDE9A)

<400> 15

aeccaaaaat actccísaacc ataaa 25

<210> 16

<211> 26

<212> DfJA

<213> Artificial Sequence

<220>

<223> Phosphatase protein 1, F regulatory pseudogene 2 (PPP1R2P2) Z subunit A region primer (inhibitor)

<400> 16

aggtttttta gçgçggaaaa aatggt 26

<210> 17

<211> 32

<212> EMA

<213> Artificial Sequence

<220>

<223> protein phosphatase 1, F regulatory pseodoaene 2 subunit 2 (pppiR2p2) A region initiator (inhibitor)

<400> 17

craaacttcc ractettaac tcaaastaao ta 32

<210> 10

<211> 32

<212> DKA

<213> Artificial sequencing <220>

<223> losfalase 1 protein, regulatory pseudogene 2 (PPR1R2P2) subunit F-region B-inflator CmibkteiO

<400> 18

gatfcttaygt ygagtagtta tttfegagtta ag

32

<21Q> Ϊ9 <211> 23 <21Z> DHA

<213> Axbificial Sequence

<220>

<223> protein phosphalase 1, R-Jnldadd PseudogeneS! (PPR1R2P2) regulatory (inhibitor)

<400> 19

saotcctcrt ccaeactccc rta 23

<210> 20 <211> 40 <212> DKA

<213> Arfeifiaial Sequence

<220>

c223> Firtitude with F-Jnldadorda FemlA Region A (C-elegans)

<210> 21 <211> 27 <212> DKA

<213> Artificial Sequence

<220>

<223> similarity of the R-end of FemiA region A (C, etegans)

<400> 21

acceaatact ccaccacztc cáaataa 27

<210> 22

<211> 27

<212> DKft

<213> Artificial Sequence

<223> similarity with F-Inhibitor of FemIA region B (C-elegane)

<400> 20

açgttaatga tttgtatatt taaaagtttt taggatattt

40

<400> 22

ayggttattt ggaygtggtg gagtatt

27

<210> 23 <2il> 30 <212> DNA

<2l3> Artificial Sequence

<220>

<223> Similaildlade with F-region A B-region primer (C. elegans} <400> 23

ccrattaacc acctccaaat taacctaata 30

<210> 24

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> CGIOO9 F-primer

<400> 24

aaaaaggygt ttggtyggtt atgagttat 29

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> R-primer CGI009

<210> 26 <211> 40 <212> DNA

<213> Artificial Sequence

<220>

<223> Carbonyl reductase 1 (CBRl) F-initiator

<400> 26

gttaygtggg tagttaatag ttagtagtta gagattagtt 40

<210> 27 <211> 26 <212> DNA

<213> Artificial Sequence

<220>

<223> R-initiator carbonyl reductase 1 (CBR1)

<210> 28 <211> 29 <212> DNA

<213> Artificial Sequence

<220>

<223> Down syndrome cell adhesion molecule (DSCAM) F-primer

<400> 25

aaactaaaat cracrtacct acaa

24

<400> 27

caaaccrata cccttattac ctccaa

26

<400> 28

ygygygttgy gtttttgtat atttgtttt

29 <210> 29 <211> 36 <212> DNA

<213> Artificial Sequence

<220>

<223> Down syndrome cell adhesion molecule R-primer (DSCAM)

<400> 29

caaaaaaaat taacaaaaaa atccatataa ctaaaa

<210> 30 <211> 33 <212> DNA

<213> Artificial Sequence

<220>

<223> Chromosome 21 open reading frame F-primer 29 (C21orf29)

<400> 30

agtttggtag ttatttgaat agttaaatga gtt

<210> 31 <211> 30 <212> DNA

<213> Artificial Sequence

<220>

<223> R-primer for chromosome 21 open reading frame 29 (C21orf29)

<400> 31

aactttctca tcctactccc taaatctata

<210> 32 <211> 26 <212> DNA

<213> Artificial Sequence

<220>

<223> F-primer CGIlll

<400> 32

tttttttagg tagttgaaag aaaagg

<210> 33 <211> 20 <212> DNA

<213> Artificial Sequence

<220>

<223> R-primer CGIlll

<400> 33

cctccctcct caaaataaac <210> 34 <211> 28 <212> DNA

<213> Artificial Sequence

<220>

<223> CGI121 F-primer

<400> 34

tttttagata ttttttgggt ttaaggtt 28

<210> 35 <211> 21 <212> DNA

<213> Artificial Sequence

<220>

<223> R-primer CGI121

<400> 35

aaatccacct acccaaacac c ??? 21

<210> 36 <211> 31 <212> DNA

<213> Artificial Sequence

<220>

<223> Region A F-primer KIAAO656

<400> 36

ttggtggtyg ygaagtgttt ttgttagtat t ??? 31

<210> 37 <211> 30 <212> DNA

<213> Artificial Sequence

<220>

<223> Region A R-primer KIAAO656

<400> 37

acctctcaaa ccraataaac ctaacaaaac 30

<210> 38 <211> 30 <212> DNA

<213> Artificial Sequence

<220>

<223> Region B F-primer KIAAO656

<400> 38

gygygygttt aayggttttg ttaggtttat 30

<210> 39 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> Region B R-Initiator KIAAO656

<400> 39 cataataata acttctcaaa cccccaatca

<210> 40

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Heat shock transcription factor 2 binding protein (HSF2BP) region A F-primer

<210> 41

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Heat shock transcription factor 2 binding protein (HSF2BP) region A R-primer

<400> 41

cracaacrac cataaacraa acra 24

<210> 42

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Heat shock transcription factor 2 binding protein (HSF2BP) B-region primer

<210> 43

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Heat shock transcription factor 2 binding protein (HSF2BP) B region R-primer

<400> 43

ttacatcaaa aactaacttt ccttctactt tacaa 35

<210> 44

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 40

tayggagtag agaagagagt gattatttat tttaygt

37

<400> 42

gtttaaatay gttggygtyg gttagggt

28

<220>

<223> Region A F-primer COL6A1 <400> 44

gttyggtygg gaggttttgt gatatt 26

<210> 45 <211> 33 <212> DNA

<213> Artificial Sequence

<220>

<223> Region A R-Primer COL6Al

<400> 45

aactacraaa craaataaac aaccrttaac ata 33

<210> 46 <211> 30 <212> DNA

<213> Artificial Sequence

<220>

<223> Region B F-primer COL6A1

<400> 46

tyggtttatt gyggttgtat tattagggtt 30

<210> 47 <211> 26 <212> DNA

<213> Artificial Sequence

<220>

<223> Region B R-primer COL6A1

<400> 47

tccataacat cgacgacact aaccaa 2 6

<210> 48 <211> 35 <212> DNA

<213> Artificial Sequence

<220>

<223> Methyl-specific CGI137 Inverse Inverter for Homogeneous MassEXTEND Protocol Primer Extension Assay

<400> 48

tctctaccta caattctata aaaaacactt atcca 35

<210> 49 <211> 17 <212> DNA

<213> Artificial Sequence

<220>

<223> Methyl-specific CGI137 extension primer for homogeneous MassEXTEND protocol primer extension assay

<400> 49

atccccaaca ttttccc

17 <210> 50 <211> 33 <212> DNA

<213> Artificial Sequence <220>

<223> Holocarboxylase synthase A (HLCS) region F-primer

<400> 50

ggagtgttaa atttggttat ttttgtttgt tat 33

<210> 51 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> Holocarboxylase synthase A (HLCS) region R-primer

<400> 51

crctaccctt ctccactaac tactcaaa 28

<210> 52 <211> 29 <212> DNA

<213> Artificial Sequence <220>

<223> Holocarboxylase Synthase (HLCS) F-region primer F

<400> 52

aggagttaga ygttttagtt ygtgtggtt 29

<210> 53 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> Holocarboxylase synthase (HLCS) B-region R-primer

<400> 53

ctaaacaccc raatccccaa aa 22

<210> 54 <211> 27 <212> DNA

<213> Artificial Sequence <220>

<223> Holocarboxylase synthase B2 (HLCS) R-primer

<400> 54

gttttagtty gtgtggttag aggtggt 27 <210> 55 <211> 31 <212> DNA

<2l3> Artificial sequence <220>

<223> R-initiator of halocarboxylase synthetase (HLCS) LC2 region

<400> 55

ctaaaaaata aaaaacaaaa tccaaaacaa a

<210> 56 <211> 28

<212> DNA

<213> Artificial sequence <220>

<223> initiator CGI009 F

<400> 56

aaaaggygtt tggtyggtta tgagttat

<210> 57 <211> 33 <212> DNA

<213> Artificial sequence <220>

<223> primer CGI009 <400> 57

aaactaaaat cracrtacct acaataccaa aaa

<210> 58 <211> 24 <212> DNA <213>

<220>

<223> primer CGI132 R <400> 58

ttgygggtta yggggattta gttt

<210> 59 <211> 24 <212> DNA

<213> artificial sequence <220>

<223> primer CGI132 F <400> 59

craaaacraa craaccaaac ctaa

<210> 60 <211> 17 <212> DNA

<213> Artificial sequence <220>

<223> Quantitative real-time PCR direct primer of halocarboxylase synthetase (HLCS)

<400> 60 ccgtgtggcc agaggtg 17

<210> 61

<211> 18

<212> DNA

<213>

<220>

<223> Quantitative real-time PCR direct primer of halocarboxylase synthetase (HLCS)

<400> 61

tgggagccgg aacctacc 18

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Real-time quantitative PCR TaqMan probe

<221> modified_base

<222> (1) ... (1)

<223> n = t modified by 6FAM

<221> modified_ base

<222> (20) ... (20)

<223> n = c modified by TAMRA

<400> 62

ncccgacctg gccctttgcn

Claims (67)

Method for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) obtaining a biological sample from the woman, in which the sample is whole blood, serum , plasma, urine or saliva; (b) determine the methylation status of a genomic sequence containing CpG in the sample, wherein the fetal genomic sequence and the female genomic sequence are differentially methylated, thereby distinguishing the female genomic sequence and the fetal genomic sequence in the sample. wherein the genomic sequence is at least 15 nucleotides in length, comprising at least one cytosine, and is within a region on chromosome 21, and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit pseudogene 2 (PPP1R2P2), FemlA similarity (Caenorhabditis elegans), CGI009, carbonyl reductase 1 (CBR1), adhesion molecule Down syndrome (DSCAM) cell, and open reading matrix 29 of chromosome 21 (C21orf29), Holocarboxylase Synthetase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; c) determine the level of the genomic sequence of the fetus; and d) comparing the level of the fetal genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy. Method according to claim 1, characterized in that the female genomic sequence is methylated and the fetal genomic sequence is not methylated. Method according to claim 1, characterized in that the female genomic sequence is not methylated and the fetal genomic sequence is methylated. Method according to claim 1, characterized in that step (b) is performed by treating the sample with a reagent that differentially modifies methylated and unmethylated DNAs. Method according to claim 4, characterized in that the reagent comprises bisulfite. Method according to claim 4, characterized in that the reagent comprises one or more enzymes which preferably cleave methylated DNA. Method according to claim 4, characterized in that the reagent comprises one or more enzymes which preferably cleave unmethylated DNA. Method according to claim 1, characterized in that step (b) is performed by methylation-specific PCR. Method for detecting or monitoring a pregnancy-associated disorder in a woman who is pregnant with a fetus, comprising the steps of: (a) obtaining DNA from a woman's biological sample, where the sample is blood integral, serum, plasma, urine or saliva; (b) treating the DNA of step (a) with bisulfite; and (c) performing an amplification reaction using the DNA of step (b) and two primers to amplify a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit 2 pseudogene 2 (inhibitor) (PPP1R2P2), Similarity to FemlA (Caenorhabditis elegans), CGI009, Carbonyl Reductase 1 (CBR1), Down Syndrome Cell Adhesion Molecule (DSCAM), Chromosome 21 Open Reading Matrix 29 (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and wherein at least one of the two primers differentially binds to the fetal genomic sequence; and d) comparing the level of the amplified portion of the genomic sequence from step (c) to a standard control, wherein an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy. Method according to claim 9, characterized in that the amplification reaction is a polymerase chain reaction (PCR). Method according to claim 9, characterized in that the amplification reaction is a methylation-specific PCR. A method according to claim 9, characterized in that the amplification reaction is an amplification based on the nucleic acid sequence. A method according to claim 9, characterized in that the amplification reaction is a filament displacement reaction. A method according to claim 9, characterized in that the amplification reaction is a branched DNA amplification reaction. Method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is preeclampsia. Method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is a premature dish. Method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is hyperemesis gravidarum. A method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is ectopic pregnancy. A method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is a chromosomal aneuploidy. Method according to claim 19, characterized in that the disorder associated with pregnancy is trisomy 21. Method according to claim 1 or 9, characterized in that the disorder associated with pregnancy is intrauterine growth retardation. 22. Method for detecting and monitoring a disorder associated with pregnancy, characterized in that it comprises the steps of: (a) obtaining DNA from a biological sample of women, in which the sample is whole blood, serum, plasma, urine. or saliva; (b) treating the DNA of step (a) with a reagent that differentially modifies methylated and unmethylated DNAs; (c) determining the nucleotide sequence of a CpG-containing genomic sequence from step (b), wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit pseudogene 2 (inhibitor) (PPP1R2P2), similarity to FemlA (Caenorhabditis elegans), CGI009, carbonyl reductase 1 (CBR1), Down Syndrome cell adhesion molecule (DSCAM), chromosome 21 open reading matrix 29 (C21 o029), Holocarboxylase Synthetase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) comparing the nucleotide sequence profile of step (c) with a standard control, wherein a change in the control pattern profile indicates the presence or progression of a disorder associated with pregnancy. Method according to claim 22, characterized in that the reagent comprises bisulfite. The method of claim 22, wherein the reagent comprises one or more enzymes which preferably cleave methylated DNA. The method of claim 22, wherein the reagent comprises one or more enzymes which preferably cleave unmethylated DNA. A method according to claim 22, further comprising an amplification step using the DNA of step (b) and two primers to amplify the genomic sequence. Method according to claim 26, characterized in that the amplification step is performed by PCR. A method according to claim 26, characterized in that the amplification step is performed by methylation-specific PCR. A method according to claim 22, characterized in that step (c) is performed by mass spectrometry. A method according to claim 22, characterized in that step (c) is performed by primer extension. Method according to claim 22, characterized in that step (c) is performed by polynucleotide hybridization. Method according to claim 22, characterized in that step (c) is performed by real time PCR. A method according to claim 22, characterized in that step (c) is performed by electrophoresis. 34. Method for detecting trisomy 21 in a fetus in a pregnant woman, comprising the steps of: (a) obtaining a biological sample from the woman, in which the sample is whole blood, serum, plasma, urine or saliva; (b) treating the sample from step (a) with a reagent that differentially modifies methylated and unmethylated DNAs; (c) analyze alleles of a CpG-containing genomic sequence, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory subunit pseudogene 2 (inhibitor) (PPP1R2P2), similarity to FemlA 0Caenorhabditis elegans), CGI009, carbonyl redutase (CBR1), Down Syndrome cell adhesion molecule (DSCAM), open reading matrix 29 of chromosome 21 (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132; and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (d) determining the ratio of alleles, wherein a deviation from that of a woman carrying a fetus having no trisomy 21 indicates trisomy - 21 in the fetus. A method according to claim 34, characterized in that the reagent comprises bisulfite. A method according to claim 34, characterized in that the reagent comprises one or more enzymes which preferably cleave methylated DNA. The method of claim 34, wherein the reagent comprises one or more enzymes which preferably cleave unmethylated DNA. A method according to claim 34, further comprising an amplification step following step (b) for amplifying the methylated and unmethylated genomic sequence. A method according to claim 38, characterized in that the amplification step is performed by PCR. A method according to claim 38, characterized in that the amplification step is performed by methylation-specific PCR. A method according to claim 34, characterized in that step (c) is performed by mass spectrometry. A method according to claim 34, characterized in that step (c) is performed by a primer extension assay. A method according to claim 34, characterized in that step (c) is performed by real time PCR. A method according to claim 34, characterized in that step (c) is performed by polynucleotide hybridization. A method according to claim 34, characterized in that step (c) is performed by electrophoresis. The method according to claim 34, characterized in that the two different alleles of the fetal genomic sequence comprise a single nucleotide polymorphism, an insert-deletion polymorphism or a single tandem repeat polymorphism. 47. A method for detecting or monitoring a disorder associated with pregnancy in a woman who is pregnant with a fetus, comprising the steps of: (a) obtaining a biological sample from the woman, in which the sample is whole blood, serum , plasma, urine or saliva; (b) determining the level of a CpG-containing genomic sequence in the sample, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one unmethylated cytosine, and is within a region on chromosome 21 and wherein the region consists of of (1) a genomic site selected from the group consisting of CGI137, phosphodiesterase 9A (PDE9A), homo sapiens protein 1 phosphatase, regulatory (inhibitor) subunit pseudogene 2 (PPP1R2P2) and FernlA (Caenorhabditis elegans) Similarity and (2) a DNA sequence of no more than 10 kb upstream and / or downstream from the site; and (c) compare the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy. 48. A method for detecting or monitoring a disorder associated with pregnancy in a woman pregnant with a fetus, comprising the steps of: (a) obtaining a biological sample from the woman, in which the sample is whole blood, serum , plasma, urine or saliva; (b) determining the level of a CpG-containing genomic sequence in the sample, wherein the genomic sequence is at least 15 nucleotides in length, comprises at least one methylated cytosine, and is within a region on chromosome 21 and wherein the region consists of (1) a genomic site selected from the group consisting of CGI009, carbonyl reductase 1 (CBR1), Down Syndrome cell adhesion molecule (DSCAM), chromosome 21 open reading matrix (C21orf29), Holocarboxylase Synthase (HLCS) and CGI132 and (2) a DNA sequence of no more than 10 kb upstream and / or downstream of the site; and (c) compare the level of the genomic sequence with a standard control, where an increase or decrease in the control pattern indicates the presence or progression of a disorder associated with pregnancy. A method according to claim 47 or 48, characterized in that step (b) comprises treating the DNA present in the blood sample with a reagent that differentially modifies methylated and unmethylated cytosine. A method according to claim 49, characterized in that the reagent comprises bisulfite. A method according to claim 49, wherein the reagent comprises one or more enzymes which preferably cleave DNA comprising methylated cytosine. The method of claim 49, wherein the reagent comprises one or more enzymes which preferably cleave DNA comprising unmethylated cytosine. A method according to claim 47 or 48, characterized in that step (b) comprises an amplification reaction. A method according to claim 53, characterized in that the amplification reaction is a polymerase chain reaction (PCR). A method according to claim 54, characterized in that the PCR is a methylation-specific PCR. A method according to claim 53, characterized in that the amplification reaction is an amplification based on the nucleic acid sequence. A method according to claim 53, characterized in that the amplification reaction is a filament displacement reaction. A method according to claim 53, characterized in that the amplification reaction is a branched DNA amplification reaction. A method according to claim 47 or 48, characterized in that the level of the genomic DNA sequence is determined by electrophoresis. A method according to claim 47 or 48, characterized in that the level of the genomic DNA sequence is determined by polynucleotide hybridization. A method according to claim 47 or 48, characterized in that the disorder associated with pregnancy is preeclampsia. Method according to claim 47 or 48, characterized in that the disorder associated with pregnancy is a premature dish. A method according to claim 47 or 48, characterized in that the disorder associated with pregnancy is hyperemesis gravidarum. A method according to claim 47 or 48, characterized in that the disorder associated with pregnancy is ectopic pregnancy. A method according to claim 47 or 48, characterized in that the disorder associated with pregnancy is trisomy 21. 66. The method of claim 47 or 48, wherein the disorder associated with pregnancy is intrauterine growth retardation. A method according to claims 1, 9, 22, 34, 47 or 48, characterized in that the genomic sequence is an island of CpG.