New fetal methylation marker

RELATED APPLICATIONS

0001This application claims priority to <patcit id="pcit0001" dnum="US79750606"><text>U.S. Provisional Patent Application Serial No. 60/797,506, filed May 3, 2006</text></patcit>, the contents of which are hereby incorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

0002Early detection of pregnancy-related conditions, including potential complications during pregnancy or delivery and genetic defects of the fetus is of crucial importance, as it allows early medical intervention necessary for the safety of both the mother and the fetus. Prenatal diagnosis has been routinely conducted using cells isolated from the fetus through procedures such as chorionic villus sampling (CVS) or amniocentesis. These conventional methods are, however, invasive and present an appreciable risk to both the mother and the fetus despite most careful handling (<nplcit id="ncit0001" npl-type="s"><text>Tabor et al., Lancet 1:1287-1293, 1986</text></nplcit>).

0003Alternatives to these invasive approaches have been developed for prenatal screening, e.g., to detecting fetal abnormalities, following the discoveries that several types of fetal cells can be found in maternal circulation (<nplcit id="ncit0002" npl-type="s"><text>Johansen et al., Prenat. Diagn. 15:921-931, 1995</text></nplcit>) and more importantly, circulating cell-free fetal DNA can be detected in maternal plasma and serum (<nplcit id="ncit0003" npl-type="s"><text>Lo et al., Lancet 350:485-487, 1997</text></nplcit>). The amount of fetal DNA in maternal blood has been shown to be sufficient for genetic analysis without complex treatment of the plasma or serum, in contrast to alternative methods requiring steps for isolating and enriching fetal cells. Fetal rhesus D (RhD) genotyping (<nplcit id="ncit0004" npl-type="s"><text>Lo et al., N. Engl. J. Med. 339:1734-1738, 1998</text></nplcit>), fetal sex determination (<nplcit id="ncit0005" npl-type="s"><text>Costa et al., N. Engl. J. Med. 346: 1502, 2002</text></nplcit>), and diagnosis of several fetal disorders (<nplcit id="ncit0006" npl-type="s"><text>Amicucci et al., Clin. Chem. 46:301-302, 2000</text></nplcit>; <nplcit id="ncit0007" npl-type="s"><text>Saito et al., Lancet 356:1170, 2000</text></nplcit>; and <nplcit id="ncit0008" npl-type="s"><text>Chiu et al., Lancet 360:998-1000, 2002</text></nplcit>) have since been achieved by detecting fetal DNA in maternal plasma or serum using a polymerase chain reaction (PCR)-based technique.

0004In addition, quantitative abnormalities of fetal DNA in maternal plasma/serum have been reported in preeclampsia (<nplcit id="ncit0009" npl-type="s"><text>Lo et al., Clin. Chem. 45: 184-188, 1999</text></nplcit> and<nplcit id="ncit0010" npl-type="s"><text> Zhong et al., Am. J. Obstet. Gynecol. 184:414-419, 2001</text></nplcit>), fetal trisomy 21 (<nplcit id="ncit0011" npl-type="s"><text>Lo et al., Clin. Chem. 45:1747-1751, 1999</text></nplcit> and <nplcit id="ncit0012" npl-type="s"><text>Zhong et al., Prenat. Diagn. 20:795-798, 2000</text></nplcit>) and hyperemesis gravidarum (<nplcit id="ncit0013" npl-type="s"><text>Sekizawa et al., Clin. Chem. 47:2164-2165, 2001</text></nplcit>). Detection of fetal nucleic acid in maternal blood for prenatal genetic analysis is also disclosed in <patcit id="pcit0002" dnum="US6258540B"><text>U.S. Patent No. 6,258,540</text></patcit>.

0005Because fetal DNA co-exists with maternal DNA in the acellular portion of a pregnant woman's blood, e.g., serum or plasma, there is a need to distinguish DNA from fetal origin and maternal origin to ensure accurate results in fetal DNA-based diagnosis. It was first disclosed in <patcit id="pcit0003" dnum="US944951"><text>U.S. Patent Application No. 09/944,951</text></patcit>, published as <patcit id="pcit0004" dnum="US20030044388A"><text>20030044388</text></patcit>, that fetal and maternal DNA may be distinguished by their different methylation profiles. <patcit id="pcit0005" dnum="US20030211522"><text>Landes et al. in U.S. Patent Application Publication No. 20030211522</text></patcit> also proposed differential methylation markers may be used for prenatal diagnosis. On the other hand, to ensure the efficacy of fetal DNA-based testing methods and to eliminate erroneous interpretation of test results due to insufficient recovery of fetal DNA obtained from such methods, there also exists a need for determining the presence and quantity of fetal DNA in a sample used for the testing procedure. It is therefore desirable to identify a fetal DNA marker that can effectively serve as a universal indicator of the presence or absence of fetal DNA in general in a test sample. It is important that such a fetal DNA marker is consistently and uniformly distinct from its maternal counterpart, and that the presence or absence of the marker can be readily determined over the background of maternal DNA and directly correlated with the presence or absence of fetal DNA in general. This invention addresses this and other related needs.

0006In this application, a number of human genes have been identified for the first time as those having highly distinct methylation patterns in fetal tissues (<i>e.g</i>., derived from placenta) and in maternal tissues. Originated from a fetus, these genes are methylated at a high level of uniformity, whereas the genes from a maternal source that releases significant amount of cell-free DNA into the maternal blood are unmethylated at a similarly high level of uniformity. These features allow the genes to effectively serve as internal positive controls of a test sample used in a prenatal diagnostic process, for the purpose of ensuring that a sufficient amount of fetal DNA has been recovered in the sample during the process. Because of the high level of uniformity in these genes' methylation status with regard to their origin, these genes are particularly reliable controls, indicative of both quality and quantity of the fetal DNA. Another advantage of these genes as fetal markers is the relative ease in detecting only the methylated fetal version in contrast to their unmethylated maternal counterparts. Furthermore, the fetal genes described in this application can also be used directly as diagnostic markers for certain conditions or disorders related to pregnancy.

BRIEF SUMMARY OF THE INVENTION

0007In the first aspect of this invention, a method is provided for detecting fetal DNA in a biological sample from a pregnant woman. This method comprises the following steps: (a) treating the sample with an agent that differentially modifies methylated and unmethylated DNA; and (b) detecting DNA sequence of <i>RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2</i> (GenBank accession No. NM_016192), or <i>PYCARD</i> (GenBank accession No. NM_013258) in the sample. The presence of the DNA sequence indicates the presence of fetal DNA in the sample, whereas the absence of the DNA sequence indicates the absence of fetal DNA in the sample.

0008In some embodiments, the sample from a pregnant woman is whole blood. In the alternative, the sample may be plasma, serum, urine, or saliva. In some embodiments, the agent capable of differentially modifying methylated and unmethylated DNA digests unmethylated DNA but not methylated DNA. This agent may be a methylation sensitive enzyme, particularly a methylation sensitive restriction enzyme, such as <i>Hpa</i> II or <i>BstU</i> I. In other embodiments, the agent may contain bisulfite.

0009In some embodiments, step (b) of the method comprises an amplification process. In an exemplary embodiment, the amplification process is a polymerase chain reaction (PCR), such as real-time PCR. In other embodiments, step (b) determines the quantity of the DNA sequence.

0010In some embodiments, when step (b) indicates the presence of fetal DNA in the sample, the method may include further steps of: (c) determining the amount of a second fetal DNA sequence in a second sample. The second sample is identical to the sample in step (a) prior to being treated with the agent that differentially modifies methylated and unmethylated DNA, and the second sequence is not <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i>; and (d) comparing the amount of the second sequence with a standard control. When an increase in the amount of the second sequence from the control is detected, it is interpreted as an indicator of either the presence of a pregnancy-associated condition or an increased risk for developing such a condition. In some cases, the second sample in step (c) is not treated with any agent that differentially modifies methylated and unmethylated DNA, whereas in other cases, the second sample in step (c) is treated with the agent before the amount of the second fetal DNA sequence is determined. For instance, the second sample in step (c) is treated with a second, different agent that differentially modifies methylated and unmethylated DNA before the amount of the second fetal DNA sequence is determined. This method is suitable for detecting the presence of various pregnancy-associated conditions or an increased risk for developing one during pregnancy. Some examples of such a condition include preeclampsia, preterm labor, and intrauterine growth retardation (IUGR).

0011In other embodiments, when step (b) indicates the presence of fetal DNA in the sample, the method may comprise the further step of: (c) detecting a second fetal DNA sequence in a second sample. The second sample is identical to the sample in step (a) prior to being treated with the agent, and the second sequence is a gene of a RhD blood type, a gene of an ABO blood type, a gene of a RhC blood type, a gene of a RhE blood type, a gene of a HLA type, or a gene located on the Y chromosome, or a gene containing a pre-determined mutation, wherein the presence of the second sequence indicates the presence of the particular RhD blood type, the particular ABO blood type, the particular RhC blood type, the particular RhE blood type, the particular HLA type, the Y chromosome, or the pre-determined mutation within the gene in the fetal genome. In some cases, the second sample in step (c) is not treated with any agent that differentially modifies methylated and unmethylated DNA. Optionally, step (c) comprises an amplification process. In an exemplary embodiment, the amplification process is a polymerase chain reaction (PCR), such as real-time PCR.

0012In the second aspect of this invention, a method is provided for detecting a pregnancy-associated condition in a pregnant women. This method comprises the following steps: (a) treating a biological sample obtained from the woman with an agent that differentially modifies methylated and unmethylated DNA; (b) detecting the amount of DNA sequence of <i>RASSF1A</i>, <i>CASP8, RARB, SCGB3Al, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i> in the sample; and (c) comparing the amount of the DNA sequence with a standard control, wherein an increase from the control indicates the presence of or an increased risk for developing the pregnancy-associated condition.

0013In some embodiments, the agent capable of differentially modifying methylated or unmethylated DNA digests unmethylated DNA but not methylated DNA. One possibility is that the agent is a methylation sensitive enzyme, such as a methylation sensitive restriction enzyme (<i>e.g</i>., <i>Hpa</i> II or <i>BstU</i> I). Another possibility is that the agent comprises bisulfite.

0014In some embodiments, step (b) of the method comprises an amplification process, which may accomplished by various means, including polymerase chain reaction (PCR), such as real-time PCR. This method is suitable for the diagnosis, monitoring, or risk assessment of a number of pregnancy-associated conditions, including is preeclampsia, preterm labor, and intrauterine growth retardation (IUGR).

BRIEF DESCRIPTION OF THE DRAWINGS

0015<ul id="ul0001" list-style="none"><li><figref idref="f0001"><b>Figure 1</b></figref><b>.</b> Primer, probe and standard calibrator sequences for <i>RASSF1A</i>, <i>APC,</i> and <i>RARB</i> sequences and PCR reaction conditions.</li><li><figref idref="f0002 f0003"><b>Figure 2</b></figref><b>.</b> Methylation status of <i>RASSF1A</i> CpG island in (a) first and (b) third trimester placental tissues and corresponding maternal blood cells. The analyzed CpG sites are numbered serially and named according to the <i>RASSF1</i> (Homo sapiens) GenBank Accession NM_007182 with the start codon of its protein coding sequence as position +1 (in parentheses). The first CpG site (-113) corresponds to chr3:50353354 (reverse strand) of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17). Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively.</li><li><figref idref="f0004 f0005"><b>Figure 3</b></figref><b>.</b> Methylation status of <i>APC</i> and <i>RARB</i> CpG islands in first- and third-trimester placental tissues and corresponding maternal blood cells. For <i>APC,</i> the analysed CpG sites are named according to GenBank Accession NM_000038 with the start codon of its protein coding sequence as position +1. The first CpG site (-17371) corresponds to chr5:112101115 of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17). For <i>RARB,</i> the analysed CpG sites are named according to GenBank Accession NM_016152 with the start codon of its protein coding sequence as position +1. The first CpG site (-73231) corresponds to chr3:25444475 (forward strand) of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17). Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively.</li><li><figref idref="f0006"><b>Figure 4</b></figref><b>.</b> Primer, probe and standard calibrator sequences for <i>CASP8</i> and PCR reaction condition.</li><li><figref idref="f0007"><b>Figure 5</b></figref><b>.</b> Methylation status of <i>CASP8</i> CpG island in first- and third-trimester placental tissues and corresponding maternal blood cells. The analysed CpG sites are named according to the <i>CASP8</i> (Homo sapiens) GenBank Accession NM_033355 with the start codon of its protein coding sequence as position +1. The first CpG site (-8167) corresponds to chr2:201948550 of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17). Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively.</li><li><figref idref="f0008"><b>Figure 6</b></figref><b>.</b> Primer, probe and standard calibrator sequences for <i>SCGB3A1</i> and PCR reaction condition.</li><li><figref idref="f0009"><b>Figure 7</b></figref><b>.</b> Methylation status of <i>SCGB3A1</i> CpG island in third-trimester placental tissues and corresponding maternal blood cells. The analysed CpG sites are named according to the SCGB3A1 (Homo sapiens) GenBank Accession NM_052863 with the start codon of its protein coding sequence as position +1. The first CpG site (-390) corresponds to chr5:179951435 of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17).</li><li><figref idref="f0010"><b>Figure 8</b></figref><b>.</b> Selective amplification of methylated DNA sequence. The circles connected to the gene sequence signify cleavage sites of methylation sensitive restriction enzyme. Open and filled circles represent unmethylated and methylated sequences, respectively, at these methylation sensitive restriction enzyme cleavage sites. The methylation sensitive restriction enzyme digestion cleaves unmethylated DNA sequence at the enzyme restriction sites. As a result, only uncleaved methylated DNA sequence could be detected in the real-time PCR amplification step.</li><li><figref idref="f0011 f0012 f0013"><b>Figure 9</b></figref><b>.</b> (a) Real-time PCR amplification plots for <i>RASSF1A</i> in placental and maternal buffy coat DNA samples. After methylation sensitive restriction enzyme digestion, <i>RASSF1A</i> sequence was detected in the placental DNA sample, but not detected in the maternal buffy coat DNA sample. (b) Real-time PCR amplification for <i>β-actin</i> sequences in placental and maternal buffy coat DNA samples. After enzyme digestion, no <i>β-actin</i> was detected in the placental or maternal buffy coat DNA. (c) Methylation status of <i>β-actin</i> CpG island in third-trimester placental tissues and corresponding maternal blood cells. The analysed CpG sites are named according to the human cytoplasmic beta-actin gene GenBank Accession M10277 with the start codon of its protein coding sequence as position +1. The first CpG site (-970) corresponds to chr7:5536879 of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17).</li><li><figref idref="f0014"><b>Figure 10.</b> (a)</figref> Schematic diagram showing the principle of the selective amplification of fetal-derived <i>RASSF1A</i> sequence in the maternal plasma DNA. The circles connected to the gene sequence signify cleavage sites of methylation sensitive restriction enzymes. Open and filled circles represent unmethylated and methylated sequences, respectively, at these methylation sensitive restriction enzyme cleavage sites. The methylation sensitive restriction enzyme digestion specifically digests unmethylated DNA at the enzyme restriction sites. As a result, maternal-derived <i>RASSF1A</i> (Rsf) and <i>β-actin</i> sequences, as well as fetal-derived <i>β-actin</i> sequence, would be digested, leaving detectable fetal-derived <i>RASSF1A</i> sequences. The filled and open arrows represent the PCR primers targeting the <i>RASSF1A</i> and <i>β-actin</i> genes, respectively. Therefore, only fetal-derived <i>RASSF1A</i> sequence could be detected by the real-time PCR system. (b) Schematic diagram illustrating the detection of incomplete enzyme digestion by the internal control of <i>β-actin</i> system. When the methylation sensitive enzyme digestion is incomplete, some maternal-derived <i>RASSF1A</i> and <i>β-actin</i> sequences, as well as some fetal derived <i>β-actin</i> sequences, would remain in the DNA sample. In this case, the <i>RASSF1A</i> signal, which may originate from both maternal-derived and fetal-derived sequences due to incomplete digestion, is not specific for fetal DNA. This internal control system is designed for minimizing the false positive detection due to incomplete enzymatic digestion.</li><li><figref idref="f0016 f0017"><b>Figure 11</b></figref><b>.</b> Real-time amplification plots for <i>RASSF1A</i> and <i>β-actin</i> for the plasma DNA samples from a third trimester pregnant woman (a) and a first trimester pregnant woman (b). After enzymatic digestion, <i>RASSF1A</i> sequence remained detectable in the maternal plasma for both women. The right shift of the amplification curve is due to the reduction in the amount of <i>RASSF1A</i> sequence after the digestion of maternal-derived <i>RASSF1A</i> sequences by the methylation sensitive restriction enzyme. In contrast, <i>β-actin</i> sequences were digested by the enzyme, and were thus not detectable. The cell-free plasma DNA samples of 71 pregnant subjects were analyzed. Twenty-eight of them were in the first trimester of their pregnancy and 43 of them were in the third trimester. <i>RASSF1A</i> sequences were detectable in <b><i>ALL</i></b> of the plasma DNA samples after methylation sensitive enzyme digestion.</li><li><figref idref="f0018"><b>Figure 12</b></figref><b>.</b> Real-time amplification plots for <i>RASSF1A</i> and <i>β-actin</i> for the maternal plasma DNA from a non-pregnant woman. After enzyme digestion, no <i>RASSF1A</i> or <i>β-actin</i> sequence was detected in the plasma DNA sample. In the 25 non-pregnant females recruited for this study, none showed detectable <i>RASSF1A</i> signal in the plasma after methylation sensitive restriction enzyme digestion.</li><li><figref idref="f0019"><b>Figure 13</b></figref><b>.</b> In this case, the <i>RASSF1A</i> genotypes of the mother (maternal buffy coat DNA) and the fetus (placental DNA) were AC and CC, respectively. Without enzyme digestion, the <i>RASSF1A</i> genotype of the maternal plasma was identical to that of the mother which was AC. After enzyme digestion, the <i>RASSF1A</i> genotype of the maternal plasma changed to CC which was identical to the placental (fetal) genotype.</li><li><figref idref="f0020"><b>Figure 14</b></figref><b>.</b> Correlations of the concentrations of <i>SRY</i> sequence vs. <i>RASSF1A</i> sequence in the maternal plasma with or without enzyme digestion for 24 third trimester pregnant women. All subjects were carrying a male fetus. There was a positive correlation between the concentrations of <i>SRY</i> and <i>RASSF1A</i> in the maternal plasma with enzyme digestion (r = 0.717, p &lt; 0.0001, Spearman correlation). However, there is no correlation between the concentrations of <i>SRY</i> and total <i>RASSF1A</i> measured without enzyme digestion (r = 0.228, p = 0.280, Spearman).</li><li><figref idref="f0021"><b>Figure 15</b></figref><b>.</b> Schematic diagram showing the strategy of non-invasive fetal rhesus D testing in pregnant women.</li><li><figref idref="f0022"><b>Figure 16</b></figref><b>.</b> The concentration of <i>RASSF1A</i> sequence after enzymatic digestion in maternal plasma was elevated in preeclamptic pregnancies.</li><li><figref idref="f0022"><b>Figure 17</b></figref><b>.</b> Primer sequences for bisulfite sequencing of <i>DAB2IP.</i></li><li><figref idref="f0023"><b>Figure 18</b></figref><b>.</b> Methylation status of <i>DAB2IP</i> CpG island in a third-trimester placental tissue sample and corresponding maternal blood cells. The analysed CpG sites are named according to the <i>DAB2IP</i> (<i>Homo sapiens</i>) GenBank Accession NM_032552 with the start codon of its protein coding sequence as position +1. The first CpG site (-59572) corresponds to chr9:121541221 of the human genome in the UCSC Genome Browser (May 2004 assembly, hg17). Filled and unfilled circles represent methylated and unmethylated CpG sites, respectively.</li></ul>

DEFINITIONS

0016The term <b>"pregnancy-associated disorder,"</b> 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 during a limited time period, e.g., during pregnancy or delivery, or may last the entire life span of the fetus following its birth. Some examples of a pregnancy-associated disorder include preeclampsia, preterm labor, and intrauterine growth retardation (IUGR).

0017In this application, the term <b>"nucleic acid"</b> or <b>"polynucleotide"</b> refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (<i>e.g</i>., degenerate codon substitutions), alleles, orthologs, mutations including point mutations, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (<nplcit id="ncit0014" npl-type="s"><text>Batzer et al., Nucleic Acid Res. 19:5081 (1991</text></nplcit>); <nplcit id="ncit0015" npl-type="s"><text>Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985</text></nplcit>); and <nplcit id="ncit0016" npl-type="s"><text>Rossolini et al., Mol. Cell. Probes 8:91-98 (1994</text></nplcit>)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

0018The term <b>"gene"</b> means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons).

0019In this application, the terms <b>"polypeptide," "peptide,"</b> and <b>"protein"</b> are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue 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 polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (<i>i.e</i>., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

0020The term <b>"amino acid"</b> refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, <i>e.g</i>., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

0021Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

0022The term <b>"bisulfite"</b> as used herein encompasses all types of bisulfites, such as sodium bisulfite, that are capable of chemically converting a cytosine (C) to a uracil (U) without chemically modifying a methylated cytosine and therefore can be used to differentially modify a DNA sequence based on the methylation status of the DNA.

0023As used herein, a reagent that <b>"differentially modifies"</b> methylated or non-methylated DNA encompasses any reagent that modifies methylated and/or unmethylated DNA in a process through which distinguishable products result from methylated and non-methylated DNA, thereby allowing the identification of the DNA methylation status. Such processes may include, but are not limited to, chemical reactions (such as a C → U conversion by bisulfite) and enzymatic treatment (such as cleavage by a methylation-dependent endonuclease). Thus, an enzyme that preferentially cleaves or digests methylated DNA is one capable of cleaving or digesting a DNA molecule at a much higher efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits a significantly higher efficiency when the DNA is not methylated.

0024In this application, the word <b>"presence"</b> or <b>"absence"</b> is used in a relative sense to describe the level of a particular DNA sequence. In other words, when a given DNA sequence or a gene is said to be "present" in a test sample, it means the level of this DNA sequence or gene is above a pre-determined threshold; whereas when a DNA sequence or gene is "absent" when its level in a test sample is below such a threshold.

0025As used in this application, an <b>"increase"</b> or a <b>"decrease"</b> refers to a detectable positive or negative change in quantity from an established standard control. An increase is a positive change preferably at least 10%, more preferably 50%, still more preferably 2-fold, even more preferably at least 5-fold, and most preferably at least 10-fold of the control value. Similarly, a decrease is a negative change preferably at least 10%, more preferably 50%, still more preferably at least 80%, and most preferably at least 90% of the control. Other terms indicating quantitative changes or differences from a comparative basis, such as <b>"more"</b> or <b>"less,"</b> are used in this application in the same fashion as described above.

0026A <b>"polynucleotide hybridization method"</b> as used herein refers to a method for detecting the presence and/or quantity 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.

0027<b>"Primers"</b> as used herein refer to oligonucleotides that can be used in an amplification method, such as a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on the polynucleotide sequence corresponding to a gene of interest, <i>e.g</i>., <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3Al, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD,</i> either methylated or unmethylated. At least one of the PCR primers for amplification of a polynucleotide sequence is sequence-specific for the sequence.

0028<b>"Standard control value"</b> as used herein refers to a predetermined amount of a genomic sequence that is originated from a fetus and is present in an established sample. The standard control value is suitable for the use of a method of the present invention, in order for comparing the amount of a gene of interest (or a non-coding sequence) that is present in a test sample. An established sample serving as a standard control provides an average amount of a fetal gene of interest that is typical for a defined time (<i>e.g</i>., first trimester) during pregnancy in the blood of an average, healthy pregnant woman carrying a normal fetus, both of whom are not at risk of developing any pregnancy-associated disorders or complications. A standard control value may vary depending on the genomic sequence of interest and the nature of the sample.

0029The term <b>"average,"</b> as used in the context of describing a pregnant woman, refers to the fact that the woman is free of at least one condition of relevance, such as a pregnancy-associated condition (<i>e.g</i>., preeclampsia or preterm labor). The term "average," when used in other context, refers to certain characteristics, such as the amount or methylation status of a particular gene of both maternal and fetal origins found in the woman's blood, that are 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 gene 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 has a similar gestational age to that of a woman whose blood is tested for indication of a potential pregnancy-associated disorder. The preferred gestational age for practicing the present invention may vary depends on the disorder that is being screened for. For example, a pregnant woman is screened for the risk of preeclampsia preferably during the second trimester of the pregnancy, whereas fetal chromosomal aneuploidy is preferably screened for and diagnosed as early as possible. Moreover, the preferred gestational age for testing may also depend on the gene of interest in testing.

0030The term <b>"preeclampsia"</b> as used herein refers to a condition that occurs during pregnancy, the main symptom of which is various forms of high blood pressure often accompanied by the presence of proteins in the urine and edema (swelling). Preeclampsia, sometimes called toxemia of pregnancy, is related to a more serious disorder called "eclampsia," which is preeclampsia together with seizures. These conditions usually develop during the second half of pregnancy (after 20 weeks), though they may develop shortly after birth or before 20 weeks of pregnancy.

0031The term <b>"preterm labor"</b> or "premature labor" 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.

0032The term <b>"intrauterine growth retardation (IUGR)"</b> refers to a condition in which the growth of the fetus is abnormally slow, its weight below the 10th percentile for gestational age. When born, the infant appears too small and undernourished for its age. IUGR, also referred to as intrauterine growth restriction, is associated with increased risk of medical illness and death in the newborn.

0033As used in this application, "a <b>gene of a RhD blood type, of an ABO blood type, of a RhC blood type, of a RhE blood type, of a HLA type, or on the Y chromosome"</b> refers to a gene that is recognized as representative of a particular blood type in accordance with the RhD, ABO, RhC, or RhE blood typing, or a particular HLA type, or a gene that is located on the Y chromosome. The detection of such a gene in fetal DNA is indicative of the fetus being a particular blood type, HLA type, or the male gender.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

0034The presence of fetal DNA in maternal plasma was first reported in 1997, offering the possibility for non-invasive prenatal diagnosis simply through the analysis of a maternal blood sample (<nplcit id="ncit0017" npl-type="s"><text>Lo et al., Lancet 350:485-487, 1997</text></nplcit>). The co-existence of fetal DNA with background maternal DNA in maternal plasma, however, demands reliable means to distinguish DNA of fetal and maternal origins. Several genes have been previously indicated as differentially methylated between their fetal and maternal versions, <i>see, e.g</i>., <nplcit id="ncit0018" npl-type="s"><text>Chim et al., Proc. Natl. Acad. Sci. USA 102:14753-14758, 2005</text></nplcit>.

0035The present inventors discovered, for the first time, that a number of genes (<i>e.g</i>., <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3Al, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> and <i>PYCARD</i>) derived from a fetus are highly methylated, whereas the same genes derived from the woman pregnant with the fetus are not methylated. Although other genes have previously been reported to have distinct methylation profile when the fetal version of the genes and maternal version are compared, the discovery by the present inventors is unique in that not only was such distinction of methylation status previously unknown with regard to these particular genes, the high level of uniformity in the methylation of the fetal genes and the lack of methylation of the maternal genes was also previously not seen. This discovery thus provides a new, more accurate, and more effective approach for distinguishing fetal and maternal genomic DNA. In particular, the detection of any one of the fetal genes identified herein in a sample during an analytic process for non-invasive prenatal diagnosis allows the confirmation that the process, including sample collection and manipulation, is operating successfully as designed in that fetal DNA in general (not limited to the genes named herein) in the sample is properly preserved both in quality and in quantity. In addition, these newly identified genes can also be used directly as fetal DNA markers to indicate the presence of or heightened risk for certain pregnancy-related conditions and complications, since these genes are uniformly methylated compared to their maternal counterparts, permitting easy distinction between the maternal copy and the fetal copy of the genes.

II. General Methodology

0036Practicing this invention utilizes routine techniques in the field of molecular biology. Basic texts disclosing the general methods of use in this invention include <nplcit id="ncit0019" npl-type="b"><text>Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001</text></nplcit>); <nplcit id="ncit0020" npl-type="s"><text>Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990</text></nplcit>); and <nplcit id="ncit0021" npl-type="b"><text>Current Protocols in Molecular Biology (Ausubel et al., eds., 1994</text></nplcit>)).

0037For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

0038Oligonucleotides that are not commercially available can be chemically synthesized, <i>e.g</i>., according to the solid phase phosphoramidite triester method first described by <nplcit id="ncit0022" npl-type="s"><text>Beaucage &amp; Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981</text></nplcit>), using an automated synthesizer, as described in <nplcit id="ncit0023" npl-type="s"><text>Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984</text></nplcit>). Purification of oligonucleotides is performed using any art-recognized strategy, <i>e.g</i>., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in <nplcit id="ncit0024" npl-type="s"><text>Pearson &amp; Reanier, J. Chrom. 255: 137-149 (1983</text></nplcit>).

0039Any one of the genes identified in the present invention, <i>RASSF1A</i>, <i>APC, CASP8, RARE, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2,</i> and <i>PYCARD,</i> and the polynucleotide sequence of synthetic oligonucleotides can be verified using, <i>e.g</i>., the chain termination method for sequencing double-stranded templates of<nplcit id="ncit0025" npl-type="s"><text> Wallace et al., Gene 16: 21-26 (1981</text></nplcit>).

III. Acquisition of Blood Samples and Extraction of DNA

0040The present invention relates to determining the presence and/or quantity of certain fetal genes found in maternal blood based on their distinct methylation status to detect the presence and/or quantity of general fetal DNA, which may be used, for example, as an internal control to indicate the proper operation of a non-invasive analytical process that utilizes fetal DNA for assessing the presence or risk of a pregnancy-associated condition or disorder. Thus, the first steps of practicing this invention are to obtain a biological sample from a pregnant woman where fetal DNA is expected to be present and treat the DNA with an agent that differentially modifies DNA based on the methylation state. One example of such an agent is one that digests only unmethylated DNA but not methylated DNA. Optionally, the DNA is first extracted from the sample.

A.

Acquisition of Blood Samples

0041A blood sample is obtained from a pregnant woman at a gestational age suitable for testing using a fetal DNA-based non-invasive diagnostic method. The suitable gestational age may vary depending on the disorder tested. Collection of blood from a woman is performed in accordance with the standard protocol hospitals or clinics generally follow. An appropriate amount of peripheral blood, <i>e.g</i>., typically between 5-50 ml, is collected and may be stored according to standard procedure prior to further preparation.

B.

Preparation of Blood Samples

0042The analysis of fetal DNA found in maternal blood according to the present invention may be performed using, <i>e.g</i>., the whole blood, serum, or plasma. The methods for preparing serum or plasma from maternal blood are well known among those of skill in the art. For example, a pregnant woman's blood can 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 through centrifugation. On the other hand, serum may be obtained with or without centrifugation following blood clotting. If centrifugation is used then it is typically, though not exclusively, conducted at an appropriate speed, <i>e.g</i>., 1,500-3,000 x g. Plasma or serum may be subjected to additional centrifugation steps before being transferred to a fresh tube for DNA extraction. In addition to the acellular portion of the whole blood, DNA may also be recovered from the cellular fraction, enriched in the buffy coat portion, which can be obtained following centrifugation of a whole blood sample from the woman and removal of the plasma.

C.

Extraction of DNA

0043There are numerous known methods for extracting DNA from a biological sample including blood. The general methods of DNA preparation (<i>e.g</i>., described by <nplcit id="ncit0026" npl-type="b"><text>Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001</text></nplcit>) can 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 GFX^™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, NJ), may 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. Methylation-Specific Chemical Modification of DNA

0044The DNA present in a sample from a pregnant woman, whether or not extracted from the sample, is then treated with an agent capable of preferentially modifying DNA depending on whether the DNA sequence is methylated. For instance, this agent can be an enzyme that digests DNA in a methylation sensitive manner, <i>i.e</i>., only unmethylated DNA will be digested while methylated DNA remains unchanged. Another possibility is that the agent selectively converts a polynucleotide sequence depending on the methylation status. Typically, such an agent reacts with the unmethylated C residue(s) in 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 allows detection and comparison of methylation status based on changes in the primary sequence of the nucleic acid. An exemplary reagent suitable for this purpose is bisulfite, such as sodium bisulfite. Methods for using bisulfite for chemical modification of DNA are well known in the art (see, <i>e.g</i>., <nplcit id="ncit0027" npl-type="s"><text>Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996</text></nplcit>) and will not be discussed in detail here.

0045As a skilled artisan will recognize, any other reagents that are unnamed here but have the same property of chemically (or through any other mechanism) modifying methylated and unmethylated DNA differentially can be used for practicing the present invention. For instance, methylation-specific modification of DNA may also be accomplished by methylation-sensitive restriction enzymes, some of which typically cleave an unmethylated DNA fragment but not a methylated DNA fragment, while others (<i>e.g</i>., methylation-dependent endonuclease McrBC) cleave DNA containing methylated cytosines but not unmethylated DNA. In addition, a combination of chemical modification and restriction enzyme treatment, <i>e.g</i>., combined bisulfite restriction analysis (COBRA), may be used for practicing the present invention.

V. Polynucleotide Sequence Amplification and Determination

0046Following the methylation-dependent differential modification of the DNA, such as chemical modification of DNA in a methylation-specific manner or methylation-sensitive enzymatic digestion, the treated DNA is then subjected to a sequence-based analysis, such that one or more of the relevant genes of the present invention (<i>e.g</i>., <i>RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i>) from the fetal source may be distinguished from their counterparts from the maternal source, and that the presence and quantity of the fetal gene(s) may be determined and compared to a standard control. Furthermore, once it is determined that one or more of these genes of fetal origin is indeed present in the sample, particularly when the amount of the gene(s) is greater than a pre-determined threshold, the sample and its equivalents are deemed to contain sufficient amount of fetal DNA for further analyses. On the other hand, one may detect and measure the quantity of these particular genes as fetal markers indicative of certain conditions or disorders related pregnancy, taking advantage of the genes' highly methylated status in contrast to the unmethylated status of their counterparts of maternal origin. For this use, the amount of one or more of the fetal genes selected from <i>RASSF1A, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> and <i>PYCARD</i> in a test sample can be compared to a standard value, where an increase from the standard value indicates the presence or heightened risk of such a pregnancy-associated disorder.

A.

Amplification of Nucleotide Sequences

0047An amplification reaction is optional prior to a sequence-based analysis for a fetal marker of this invention after treatment by the methylation-dependent differential modification process. In some embodiments of this invention, the amplification is performed to preferentially amplify a fetal marker of this invention that has a particular methylation pattern, such that only the genomic sequence from one particular source, <i>e.g</i>., from the placenta or other tissues of the fetus, is detected and analyzed.

0048A variety of polynucleotide amplification methods are well established and frequently used in research. For instance, the general methods of polymerase chain reaction (PCR) for polynucleotide sequence amplification are well known in the art and are thus not described in detail herein. For a review of PCR methods, protocols, and principles in designing primers, <i>see, e.g</i>., <nplcit id="ncit0028" npl-type="b"><text>Innis, et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc. N.Y., 1990</text></nplcit>. PCR reagents and protocols are also available from commercial vendors, such as Roche Molecular Systems.

0049PCR is most usually carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available.

0050Although PCR amplification of a target polynucleotide sequence (<i>e.g</i>., that of <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i>) is typically used in practicing the present invention, one of skill in the art will recognize that the amplification of a genomic sequence found in a maternal blood sample may be accomplished by any known method, such as ligase chain reaction (LCR), transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA), each of which provides sufficient amplification. More recently developed branched-DNA technology may also be used to qualitatively demonstrate the presence of a particular genomic sequence of this invention, which represents a particular methylation pattern, or to quantitatively determine the amount of this particular genomic sequence in the maternal blood. For a review of branched-DNA signal amplification for direct quantification of nucleic acid sequences in clinical samples, see<nplcit id="ncit0029" npl-type="s"><text> Nolte, Adv. Clin. Chem. 33:201-235, 1998</text></nplcit>.

B.

Determination of Polynucleotide Sequences

0051Techniques for polynucleotide sequence determination are also well established and widely practiced in the relevant research field. For instance, the basic principles and general techniques for polynucleotide sequencing are described in various research reports and treatises on molecular biology and recombinant genetics, such as Wallace <i>et al</i>., <i>supra</i>; Sambrook and Russell, <i>supra,</i> and Ausubel <i>et al</i>., <i>supra.</i> DNA sequencing methods routinely practiced in research laboratories, either manual or automated, can be used for practicing the present invention. Additional means suitable for detecting changes (<i>e.g</i>., 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.

VI. Establishing a Standard Control Value

0052In order to establish a standard control value for practicing the method of this invention, a group of healthy pregnant women carrying healthy fetuses are first selected. These women are of similar gestational age, which is within the appropriate time period of pregnancy for screening of conditions such as preeclampsia and preterm labor using the methods of the present invention.

0053The healthy status of the selected pregnant women and the fetuses they are carrying are confirmed by well established, routinely employed methods including but not limited to monitoring blood pressure of the women, recording the onset of labor, and conducting fetal genetic analysis using CVS and amniocentesis.

0054Furthermore, the selected group of healthy pregnant women carrying healthy fetuses must be of a reasonable size, such that the average amount of a particular fetal gene identified in this invention present in the maternal blood obtained from the group can be reasonably regarded as 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.

0055Once an average level is established for a particular fetal gene present in the maternal blood based on the individual values found in each woman of the selected healthy control group, this average or median or representative value is considered a standard control value. Any biological sample (<i>e.g</i>., a blood sample) that contains a similar amount of the fetal gene can thus be used to provide a standard control value for samples of the same kind (<i>e.g</i>., blood samples). Furthermore, a solution containing a genomic DNA sequence in the average or median or representative amount can also be artificially assembled and to provide a standard control value. Standard control value may differ from gene to gene and depending on the nature of biological samples, <i>i.e</i>., the standard control value for <i>RASSF1A</i> may be different for a plasma sample from that for a saliva sample.

VII

.

Summary of invention

0056The invention is summarized as follows: <ol id="ol0001" compact="compact"><li>1. A method for detecting fetal DNA in a biological sample from a pregnant woman, comprising the steps of: <ol id="ol0002" compact="compact"><li>(a) treating the sample with an agent that differentially modifies methylated and unmethylated DNA; and</li><li>(b) detecting DNA sequence of <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3Al, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i> in the sample, wherein the presence of the DNA sequence indicates the presence of fetal DNA in the sample, and the absence of the DNA sequence indicates the absence of fetal DNA in the sample.</li></ol></li><li>2. The method of item 1, wherein the sample is whole blood.</li><li>3. The method of item 1, wherein the sample is plasma.</li><li>4. The method of item 1, wherein the sample is serum.</li><li>5. The method of item 1, wherein the sample is urine.</li><li>6. The method of item 1, wherein the sample is saliva.</li><li>7. The method of item 1, wherein the agent digests unmethylated DNA but not methylated DNA.</li><li>8. The method of item 7, wherein the agent is a methylation sensitive enzyme.</li><li>9. The method of item 8, wherein the methylation sensitive enzyme is a methylation sensitive restriction enzyme.</li><li>10. The method of item 9, wherein the methylation sensitive restriction enzyme is <i>Hpa</i> II or <i>BstU</i> I.</li><li>11. The method of item 1, wherein the agent comprises bisulfite.</li><li>12. The method of item 1, wherein step (b) comprises an amplification process.</li><li>13. The method of item 12, wherein the amplification process is a polymerase chain reaction (PCR).</li><li>14. The method of item 13, wherein the PCR is real-time PCR.</li><li>15. The method of item 1, wherein step (b) determines the amount of the DNA sequence.</li><li>16. The method of item 1, wherein step (b) indicates the presence of fetal DNA in the sample, the method further comprising the steps of: <ul id="ul0002" list-style="none" compact="compact"><li>(c) determining the amount of a second fetal DNA sequence in a second sample, wherein the second sample is identical to the sample in step (a) prior to being treated with the agent, and the second sequence is not <i>RASSF1A</i>, <i>APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i>; and</li><li>(d) comparing the amount of the second sequence with a standard control, wherein an increase in the amount of the second sequence from the control indicates the presence or an increased risk for developing a pregnancy-associated condition.</li></ul></li><li>17. The method of item 16, wherein the second sample in step (c) is not treated with any agent that differentially modifies methylated and unmethylated DNA.</li><li>18. The method of item 16, wherein the second sample in step (c) is treated with the agent before the amount of the second fetal DNA sequence is determined.</li><li>19. The method of item 16, wherein the second sample in step (c) is treated with a second, different agent that differentially modifies methylated and unmethylated DNA before the amount of the second fetal DNA sequence is determined.</li><li>20. The method of item 16, wherein the pregnancy-associated condition is preeclampsia, preterm labor, or intrauterine growth retardation (IUGR).</li><li>21. The method of item 1, wherein step (b) indicates the presence of fetal DNA in the sample, the method further comprising the step of: (c) detecting a second fetal DNA sequence in a second sample, wherein the second sample is identical to the sample in step (a) prior to being treated with the agent, and the second sequence is a gene of a RhD blood type, of an ABO blood type, of a RhC blood type, of a RhE blood type, of a HLA type, on the Y chromosome, or containing a pre-determined mutation, wherein the presence of the second sequence indicates the presence of the RhD blood type, the ABO blood type, the RhC blood type, the RhE blood type, the HLA type, the Y chromosome, or the mutation in the fetal genome.</li><li>22. The method of item 21, wherein the second sample in step (c) is not treated with any agent that differentially modifies methylated and unmethylated DNA.</li><li>23. The method of item 21, wherein step (c) comprises an amplification process.</li><li>24. The method of item 23, wherein the amplification process is a polymerase chain reaction (PCR).</li><li>25. The method of item 24, wherein the PCR is real-time PCR.</li><li>26. A method for detecting a pregnancy-associated condition in a pregnant women, comprising the steps of: <ol id="ol0003" compact="compact"><li>(a) treating a biological sample obtained from the woman with an agent that differentially modifies methylated and unmethylated DNA;</li><li>(b) detecting the amount of DNA sequence of <i>RASSF1A</i>, <i>CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1</i>, <i>TMEFF2,</i> or <i>PYCARD</i> in the sample; and</li><li>(c) comparing the amount of the DNA sequence with a standard control, wherein an increase from the control indicates the presence or an increased risk for developing the pregnancy-associated condition.</li></ol></li><li>27. The method of item 26, wherein the agent digests unmethylated DNA but not methylated DNA.</li><li>28. The method of item 27, wherein the agent is a methylation sensitive enzyme.</li><li>29. The method of item 28, wherein the methylation sensitive enzyme is a methylation sensitive restriction enzyme.</li><li>30. The method of item 29, wherein the methylation sensitive restriction enzyme is <i>Hpa</i> II or <i>BstU</i> I.</li><li>31. The method of item 26, wherein the agent comprises bisulfite.</li><li>32. The method of item 26, wherein step (b) comprises an amplification process.</li><li>33. The method of item 32, wherein the amplification process is a polymerase chain reaction (PCR).</li><li>34. The method of item 33, wherein the PCR is real-time PCR.</li><li>35. The method of item 26, wherein the pregnancy-associated condition is preeclampsia, preterm labor, or intrauterine growth retardation (IUGR).</li></ol>

EXAMPLES

0057The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1: Tumor suppressor genes that are hypermethylated in the fetus compared with maternal blood

0058We aimed to identify epigenetic markers that are fetal-specific in maternal blood. Previous data suggest that fetal DNA molecules in maternal plasma are predominantly derived from the placenta (<nplcit id="ncit0030" npl-type="s"><text>Chim et al., Proc Natl Acad Sci US A.,102, 14753-14758</text></nplcit>; <nplcit id="ncit0031" npl-type="s"><text>Masuzaki et al., J Med Genet 41, 289-292, 2004</text></nplcit>; <nplcit id="ncit0032" npl-type="s"><text>Flori et al., Hum Reprod 19, 723-724, 2004</text></nplcit>), while the background DNA in maternal plasma may originate from maternal blood cells (<nplcit id="ncit0033" npl-type="s"><text>Lui et al., Clin Chem 48, 421-427, 2002</text></nplcit>). Hence, to identify fetal epigenetic markers, the methylation profiles of genomic loci were assessed in both placental tissues and maternal blood cells with an aim to identify loci that demonstrate differential methylation between the two tissue types. Such markers can be used for prenatal diagnosis and monitoring of pregnancy-related conditions.

<i>Subject Recruitment and Sample Collection</i>

0059Subjects were recruited from the Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong. The study and the collection of human clinical samples were approved by the institutional review board. Informed consent was sought from each subject. First trimester placental tissues were collected immediately after elective pregnancy terminations.

0060Third trimester placental tissues were collected after elective cesarean delivery of uncomplicated pregnancies. Maternal peripheral blood samples (12 mL EDTA) were collected just prior to the performance of obstetrics procedures.

<i>Sample Processing and Bisulfite Sequencing</i>

0061Blood samples were centrifuged at 1,600g for 10 min at 4°C. After the removal of the supernatant, the peripheral blood cell portion was re-centrifuged at 2,500g. Any residual plasma was further removed. DNA was extracted from peripheral blood cells using the Nucleon Blood DNA extraction kit (GE Healthcare-Biosciences, Little Chalfont, United Kingdom) and from placental tissues using the QIAamp Tissue Kit (Qiagen, Hilden, Germany), each according to the manufacturer's instructions. Bisulfite converts unmethylated cytosine into uracil, while leaving methylated cytosine unchanged. Extracted DNA samples were bisulfite converted using the CpGenome Universal DNA Modification Kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. For each conversion reaction, 1 µg of DNA was incubated at 50°C for 16 hours after the addition of Reagent I. Each bisulfite converted DNA sample was subjected to PCR by primers that did not discriminate between methylated and unmethylated sequences, using a GeneAmp PCR Core Reagent kit (Applied Biosystems). Each subsequent PCR product was TA-cloned into pGEM-Teasy vector (Promega) for transformation into <i>E. coli</i> strain JM109, according to the manufacturer's instructions. Clones were picked randomly and colony PCR was then performed using vector primers T7 and SP6 to amplify the cloned inserts. Cycle sequencing was performed using BigDye version 1.1 (Applied Biosystems) and an automated capillary DNA sequencer Genetic Analyzer 3100 (Applied Biosystems). The sequences obtained were aligned and compared using SeqScape software (Applied Biosystems). The completeness of bisulfite conversion was first confirmed before scoring. The CpG sites sequenced as cytosine or thymine residues were scored as methylated or unmethylated, respectively. The methylated site frequency was calculated for each sample by dividing the total number of methylated sites over all cloned CpG sites.

<i>Data Comparison and Statistical Analysis</i>

0062Following bisulfite conversion, a CpG site was scored as methylated if the sequence was cytosine; scored as unmethylated if it was occupied by a thymine residue (deoxy counterpart of uracil). The methylated site frequency was calculated for each sample by dividing the total number of methylated sites over all cloned CpG sites.

RASSF1A

,

APC,

and

RARE

0063PCR primers for bisulfite sequencing and the PCR cycling conditions are listed in <figref idref="f0001">Figure 1</figref>. The bisulfite sequencing results are listed in <figref idref="f0002 f0003">Figures 2</figref> and <figref idref="f0004 f0005">3</figref>. These results indicated that <i>RASSF1A</i>, <i>APC,</i> and <i>RARB</i> sequences were hypermethylated in the placenta, but not methylated in the maternal blood cells.

CASP8

0064The PCR primers for bisulfite sequencing and PCR cycling conditions are listed in <figref idref="f0006">Figure 4</figref>. Bisulfite sequencing results are listed in <figref idref="f0007">Figure 5</figref>. These results indicated that <i>CASP8</i> sequences were hypermethylated in the placenta, but not methylated in the maternal blood cells.

SCGB3A1

0065The PCR primers for bisulfite sequencing and PCR cycling conditions are listed in <figref idref="f0008">Figure 6</figref> Bisulfite sequencing results are listed in <figref idref="f0009">Figure 7</figref>. These results indicated that <i>SCGB3A1</i> sequences were hypermethylated in the placenta, but largely not methylated in the maternal blood cells.

DAB2 interacting protein (DAB2IP)

0066The PCR primers for bisulfite sequencing and PCR cycling conditions are listed in <figref idref="f0022">Figure 17</figref>. Bisulfite sequencing results for one placental tissue sample and the corresponding maternal blood cells are listed in <figref idref="f0023">Figure 18</figref>. These results indicated that <i>DAB2IP</i> sequences were hypermethylated in the placenta, but not methylated in the maternal blood cells.

Example 2: Methylation Sensitive Enzyme Digestion Followed by Real-Time PCR

0067Methylation sensitive enzymes are enzymes that cut DNA only when their recognition site is not methylated. For more description, <i>see, e.g</i>., U.S. patent application publication No. <patcit id="pcit0006" dnum="US20050158739A"><text>US 2005/0158739 (Jeddeloh et al.</text></patcit>)<i>.</i> For example, <i>BstU</i> I recognizes the CGCG site and cuts the DNA when the CpG is not methylated. As shown in <figref idref="f0010">Figure 8</figref>, more than one methylation sensitive enzymes can be used to digest unmethylated DNA, leaving only the methylated sequences intact. Methods such as real-time PCR can be used subsequently to detect the DNA sequences which are still amplifiable after enzymatic digestion.

MATERIALS AND METHODS

<i>Subject Recruitment and Sample Collection</i>

0068Subjects were recruited from the Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong. The study and the collection of human clinical samples were approved by the institutional review board. Informed consent was sought from each subject. Third trimester placental tissues were collected after elective cesarean delivery of uncomplicated pregnancies. Maternal peripheral blood samples were collected just prior to the performance of obstetric procedures.

<i>Sample Processing and DNA Digestion by Methylation Sensitive Enzyme</i>

0069Maternal blood samples were centrifuged at 1,600g for 10 min and at 16,000g for 10 min at 4°C. DNA was extracted from the 200 µl of buffy coat and 0.2 g of placental tissue using the QIAamp DNA Blood mini kit and the QIAamp DNA mini kit, respectively, following the manufacturer's recommendation. One hundred nanograms of placental and maternal buffy coat DNA were digested with 100U of <i>Bst</i>U I, a methylation sensitive enzyme, in 1X digestion buffer at 60°C for 16 hours.

<i>Real-Time PCR Detection for RASSF1A Sequence</i>

0070<i>RASSF1A</i> sequence was amplified by real-time PCR using the primers RSF-b151F, 5'-AGCCTGAGCTCATTGAGCTG-3' and RSF-dsgnR, 5'-ACCAGCTGCCGTGTGG-3', and the minor grove binding (MGB) fluorescent probe RSF-dsgnT, 5'-FAM-CCAACGCGCTGCGCAT-MGB-3'. The timing of or the number of PCR cycles required for the appearance of detectable fluorescent signal is inversely correlated with the amount of <i>RASSF1A</i> sequence in the input DNA sample. In other words, the higher the amount of <i>RASSF1A</i> sequence present in a DNA sample, the earlier the fluorescent signal would appear, resulting in a lower threshold cycle number.

0071<figref idref="f0011">Figure 9a</figref> shows an example of real-time PCR quantification of <i>RASSF1A</i> molecules from the placenta and maternal blood cells after <i>BstU</i> I digestion. Without enzyme digestion, <i>RASSF1A</i> molecules from both the placenta and maternal buffy coat were detected. With enzyme digestion, only <i>RASSF1A</i> molecules from the placenta were detected. For <i>β-actin</i> molecules, they were detectable only without enzyme digestion, regardless of their origins (<figref idref="f0012">Figure 9b</figref>). It is expected since the <i>β-actin</i> sequence is not methylated. <figref idref="f0013">Figure 9c</figref> shows the bisulfite sequencing results of the <i>β-actin</i> gene for the placenta and maternal buffy coat. Both the placenta tissues and maternal buffy coat were completely unmethylated.

Example 3: Detection of <i>RASSF1A</i> in maternal plasma after enzymatic digestion

0072In example 1, by bisulfite sequencing, we have demonstrated that the <i>RASSF1A</i> gene of maternal blood cells is completely unmethylated while that of the placenta (of fetal origin) is heavily methylated. In example 2, we have demonstrated that the <i>RASSF1A</i> sequences from maternal blood cells were completely digested by a methylation sensitive restriction enzyme, while the <i>RASSF1A</i> sequences from placenta were only partially digested by the same enzyme. Plasma DNA is composed of DNA of maternal origin (largely from maternal blood cells and thus is unmethylated) and DNA of fetal origin (placenta being a main contributor and thus is methylated for the markers described in this patent application). It is thus feasible to use methylation sensitive enzyme digestion of plasma DNA to remove the maternal DNA background and to increase the fractional concentration of the fetal-derived <i>RASSF1A</i> molecules in maternal plasma.

0073A schematic diagram is shown in <figref idref="f0014">Figure 10a</figref>, maternal-derived <i>RASSF1A</i> is unmethylated and thus is digested by methylation sensitive enzyme while some of the fetal-derived <i>RASSF1A</i> is methylated and thus is not digested. If enzyme digestion is complete, only fetal-derived, methylated DNA is left intact and can serve as the template for PCR amplification. In the case of <i>β-actin,</i> since both the maternal and fetal-derived DNA sequences are unmethylated, complete enzyme digestion will destroy all sequences and subsequently no PCR amplification using this <i>β-actin</i> sequence can be achieved. Human errors and reagent quality sometimes may cause incomplete enzyme digestion. <i>Beta-actin</i> can thus be used as an internal control to indicate incomplete digestion (<figref idref="f0015">Figure 10b</figref>). If PCR amplification is successful for <i>β-actin</i> after enzyme digestion, it is likely that the enzyme digestion is incomplete. In this case, the entire assay may need to be repeated until a negative result is achieved for the <i>β-actin</i> assay.

MATERIALS AND METHODS

Sample Processing and DNA Digestion by Methylation Sensitive Enzyme

0074Maternal blood samples were centrifuged at 1,600g for 10 min and at 16,000g for 10 min at 4°C. DNA was extracted from 1.6 ml plasma using the QIAamp mini kit (Qiagen) and eluted with 50 µl of H₂O. Thirty-five microliters of plasma DNA were digested with 100U of <i>BstU</i> I enzyme, in 1X digestion buffer at 60°C for 16 hours.

<i>Real- Time PCR Detection for RASSF1A and Beta-Actin Sequences</i>

0075<i>RASSF1A</i> sequence was amplified and quantified by real-time PCR as described above. <i>Beta-actin</i> sequence was amplified and quantified by real-time PCR using the primers Actin-163F, 5'-GCGCCGTTCCGAAAGTT-3' and Actin-298R, 5'-CGGCGGATCGGCAAA-3', and the MGB fluorescent probe Actin-243T, 5'-VIC-ACCGCCGAGACCGCGTC-MGB-3'. <i>SRY</i> sequence was amplified and quantified using the primers <i>SRY</i>-109F, 5'-TGGCGATTAAGTCAAATTCGC-3' and <i>SRY</i>-245R, 5'-CCCCCTAGTACCCTGACAATGTATT-3', and the fluorescent probe <i>SRY</i>-142T, 5'-FAM-AGCAGTAGAGCAGTCAGGGAGGCAGA-TAMRA-3'. A DNA construct containing one copy each of the <i>RASSF1A</i>, <i>SRY</i> and <i>β-actin</i> amplicons was established as the quantitative standard of the three assays. A calibration curve was created by serial dilutions of a known quantity of the DNA construct and was included in each round of real-time PCR for the quantification of plasma <i>RASSFIA, β-actin</i> and <i>SRY.</i>

0076<figref idref="f0016 f0017">Figure 11</figref> shows an example of real-time PCR quantification of <i>RASSF1A</i> molecules from 1^st and 3^rd trimester maternal plasma with and without <i>BstU</i> I digestion. For the 1st trimester sample, <i>RASSF1A</i> concentration was reduced from 688 copies/mL plasma to 49 copies/mL plasma due to enzyme digestion. This dramatic reduction is expected since the majority (on average 96.6%, <nplcit id="ncit0034" npl-type="s"><text>Lo et al., Am J Hum Genet 1998, 62: 768-775</text></nplcit>) of the DNA molecules are of maternal origin, and thus are unmethylated and digested. For the third trimester sample, <i>RASSF1A</i> concentration was reduced from 1275 copies/mL plasma to 74 copies/mL plasma due to enzyme digestion. We have analyzed 71 pregnant women (28 in 1^st trimester and 43 in 3^rd trimester) using this assay on <i>RASSF1A</i> molecules. <i>RASSF1A</i> sequences were detectable in all of the plasma DNA samples after methylation sensitive enzyme digestion. <i>Beta-actin</i> digestion control was analyzed for all the samples. For every case, <i>β-actin</i> sequence was detected only without enzyme digestion.

Example 4: Demonstration of Fetal-Specificity of <i>RASSF1A</i> Sequence in Maternal Plasma after Enzymatic Digestion

0077Fetal-specificity of the DNA sequences after enzymatic digestion is important for the application to prenatal diagnosis and pregnancy monitoring. In this example, we shall demonstrate the fetal-specificity of <i>RASSF1A</i> sequence after enzymatic digestion in maternal plasma by four lines of experiments.

0078In the first experiment, it is demonstrated that the <i>RASSF1A</i> sequence was not detectable after enzymatic digestion in non-pregnant women. This is important since if the <i>RASSF1A</i> molecules after enzymatic digestion are fetal-specific, they should not be detected in non-pregnant women. In <figref idref="f0018">Figure 12</figref>, one case of non-pregnant women is shown. <i>RASSF1A</i> sequence was only detected without enzyme digestion. With enzyme digestion, no detectable <i>RASSF1A</i> sequence was found. As expected, the <i>β-actin</i> digestion control was only detected without enzyme digestion. In the 25 non-pregnant volunteers we have recruited for this study, none of them showed detectable <i>RASSF1A</i> signal in the plasma after methylation sensitive restriction enzyme digestion.

0079In the second experiment, it is demonstrated that the <i>RASSF1A</i> sequence after enzymatic digestion was not detectable after the delivery of the baby. If the <i>RASSF1A</i> molecules after enzymatic digestion are fetal specific, it is expected that they will disappear from the maternal plasma since the sources (placenta being a main one) that release such molecules are removed after delivery. Other fetal specific markers such as <i>SRY</i> had been shown to demonstrate similar clearance after delivery (<nplcit id="ncit0035" npl-type="s"><text>Lo et al., Am J Hum Genet 1998, 62: 768-775</text></nplcit>). Five pairs of pre- and post-delivery maternal plasma samples were collected. For all cases, after enzymatic digestion, <i>RASSF1A</i> molecules were detectable before delivery, but not detectable after delivery (Table 1). Similarly, the fetal-specific <i>SRY</i> marker was only detectable before delivery.

0080In the third experiment, a single nucleotide polymorphism (SNP) marker was used to distinguish the maternal-derived and fetal-derived <i>RASSF1A</i> sequences. If the <i>RASSF1A</i> sequences after enzymatic digestion are indeed fetal specific, then the genotype of such sequences should be that of the fetus, instead of that of the mother. For example, if a SNP marker is CC in the fetus and AC in the mother, the genotype of the DNA after enzymatic digestion should be CC, instead of AC. Similarly, for an AC/CC (fetal/maternal) pair, the genotype of the DNA after enzymatic digestion should be AC, instead of CC. For the plasma DNA without digestion, the genotype may be the same as that of the mother, since the majority of the plasma DNA is of maternal origin.

MATERIALS AND METHODS

<i>RASSF1A</i> Genotyping

0081DNA was extracted from maternal plasma, maternal buffy coat and placenta as described above. Thirty-five microliters of each maternal plasma DNA sample were subjected to <i>BstU</i> I enzyme digestion for 16 hours as described above. PCR amplification of the <i>RASSF1A</i> sequence was performed with the primers RSF-b151F, 5'-AGCCTGAGCTCATTGAGCTG-3' and RSF-dsgnR, 5'-ACCAGCTGCCGTGTGG-3' using maternal buffy coat DNA, placental DNA, maternal plasma DNA without enzymatic digestion and maternal plasma DNA after enzymatic digestion as templates. As there is a single nucleotide polymorphism (SNP id: rs4688725) within this <i>RASSF1A</i> amplicon, the <i>RASSF1A</i> genotypes of different tissues may be determined. A primer extension reaction was set up for the genotyping of <i>RASSF1A</i> DNA. Each 14 µl reaction contained 10 µl of PCR products, 0.77 µM extension primer Rsf-R17 5'-CAGCCGGGTGGGCCCT-3', 1.15 U thermosequenase and a mixture of dideoxynucleotides (ddATP, ddCTP and ddTTP) and the deoxynucleotide dGTP (64 µM each). For <i>RASSF1A</i> sequence with a genotype A, the primer would be extended to produce 5'-CAGCCGGGTGGGCCCTddT-3' with a molecular weight of 5476.6 Da. For <i>RASSF1A</i> sequence with a genotype C, the primer would be extended to produce 5'-CAGCCGGGTGGGCCCTGddC-3' with a molecular weight of 5790.8 Da. The final base extension products were analyzed by the MassARRAY MALDI-TOF mass spectrometry (SEQUENOM). The genotype of the <i>RASSF1A</i> was determined by the TyperAnalyzer software (SEQUENOM).

0082<figref idref="f0019">Figure 13</figref> shows an example of the genotyping experiment result for maternal plasma DNA with or without enzyme digestion, maternal buffy coat DNA and placental DNA. As expected, the genotype of the maternal plasma DNA after enzymatic digestion was the same as that of the placenta (fetal genotype), but not that of the maternal buffy coat. Table 2 shows the genotyping results for 43 cases where maternal plasma DNA with or without enzyme digestion, maternal buffy coat DNA and placental DNA were all analyzed. In each of the 43 cases, the genotype of the maternal plasma DNA after enzymatic digestion was identical to the placental (fetal) genotype.

0083In the fourth experiment, we demonstrated that two markers, namely the <i>RASSF1A</i> sequence after enzymatic digestion and <i>SRY</i> sequence, had concentrations in the maternal plasma that correlated with each other. Plasma DNA was extracted from 24 3^rd trimester pregnant women carrying a single male fetus. As shown in <figref idref="f0020">Figure 14</figref>, a positive correlation was observed between <i>RASSF1A</i> sequence after enzymatic digestion and <i>SRY</i> sequence (r = 0.717, p &lt; 0.0001, Spearman correlation). Additionally, the concentration of total <i>RASSF1A</i> without enzymatic digestion, which was derived predominantly from the mother, did not correlate with that for <i>SRY.</i>

0084These four experiments demonstrated, conclusively, that the <i>RASSF1A</i> sequence after enzymatic digestion was exclusively (to the extent of the techniques we used) of fetal origin. <i>RASSF1A</i> is thus useful for prenatal diagnosis and pregnancy monitoring.

Example 5: Demonstration of <i>RASSF1A</i> as A Positive Analytical Marker for Prenatal Diagnosis of RhD Blood Type

0085Rhesus D (RhD) blood group incompatibility is an important cause of hemolytic disease of the fetus and newborn. The pathogenesis of this disorder involves alloimmunization of a RhD negative pregnant woman by RhD antigen encoded by the paternal allele and displayed on the surface of fetal red cells. Maternal alloimmunization usually occurs during delivery when the tissue of a RhD positive fetus comes into contact with maternal blood. This would generate anti-RhD antibodies that can cross the placenta and destroy fetal red cells in the subsequent pregnancies with a RhD positive fetus. Maternal alloimmunization can be prevented or minimized by giving prophylactic anti-RhD immunoglobulin before and after the delivery of the first RhD positive baby. Therefore, it is beneficial to know the RhD status of a fetus before delivery. However, obtaining fetal cells for RhD genotyping/phenotyping by amniocentesis carries a risk of transplacental hemorrhage, which, if the fetus is RhD positive, could sensitize the maternal production of anti-RhD. The development of non-invasive prenatal RhD genotyping offers a safe alternative to obtaining fetal cells for RhD genotyping (<nplcit id="ncit0036" npl-type="s"><text>Lo et al., N Engl J Med 1998; 339: 1734-1738</text></nplcit>). This technique involves the detection of fetal RhD sequence in the maternal plasma. The presence of RhD sequence in the plasma of a RhD negative pregnant women would indicate a RhD positive fetus. However, the absence of such sequence in the maternal plasma can be interpreted in two ways: 1) the fetus is RhD negative; or 2) there is inadequate fetal DNA in the maternal plasma to allow accurate fetal RhD typing. A universal fetal DNA marker as a positive control in the maternal plasma DNA would be useful to exclude the second possibility. The detection of the positive control fetal DNA marker in maternal plasma DNA would support the presence of a RhD negative fetus while the absence of the fetal DNA marker would suggest inadequate fetal DNA in maternal plasma. To date, available fetal DNA markers that can be used as positive control include Y chromosomal DNA and DNA polymorphisms. Both of these two types of markers are only applicable to a subset of pregnancies. Y chromosomal DNA is only applicable to pregnancies with a male fetus. DNA polymorphism is only applicable to particular genotype combinations where certain genotype is present only in the fetus, but not in the pregnant woman. In this regard, the methylated <i>RASSF1A</i> sequence illustrated in the above sections could be used as a universal fetal DNA marker that is applicable to all pregnancies regardless of the gender or polymorphism of the fetus. Those of skill in the art will also recognize that, besides the methylated <i>RASSF1A</i> marker, the other markers described herein could also be used in such a fashion.

0086<figref idref="f0021">Figure 15</figref> shows a schematic diagram outlining one strategy for non-invasive RhD typing of the fetus. This is by no means the only way that a hypermethylated fetal DNA marker can be used as a positive analytical marker for prenatal diagnosis of RhD blood type. Those of skill in the art can also appreciate that a hypermethylated fetal DNA marker such as <i>RASSF1A</i> can be used as a positive analytical marker for prenatal diagnosis of other conditions, such as β-thalassemia, cystic fibrosis, congenital adrenal hyperplasia, and chromosomal aneuploidies. This positive analytical marker may also be assessed prior to, or simultaneously with the actual prenatal assessment of a condition such as RhD blood type.

MATERIALS AND METHODS

<i>Subject Recruitment and Sample Collection</i>

0087Subjects undergoing first trimester Down syndrome screening were recruited from the King's College Hospital London, United Kingdom. The study and the collection of human clinical samples were approved by the institutional review board. Informed consent was sought from each subject. Chorionic villus tissues were collected via chorionic villus sampling procedure. Maternal peripheral blood samples were collected just prior to the performance of obstetrics procedures.

<i>Sample Processing and Real-Time PCR Detection for RHD Sequences</i>

0088DNA was extracted from maternal plasma, buffy coat and CVS samples as described in previous sections. The RhD status of the mother and the fetus were determined by real-time amplification of a sequence on the exon 7 and exon 10 of the <i>RHD</i> gene as described previously (<nplcit id="ncit0037" npl-type="s"><text>Lo et al., N Engl J Med 1998, 339: 1734-1738</text></nplcit>; <nplcit id="ncit0038" npl-type="s"><text>Rijnders et al., Obstet Gynecol 2004, 103:157-164</text></nplcit>) using maternal buffy coat DNA and CVS DNA as templates, respectively. In our cohort, the exon 7 and exon 10 assays gave identical results for all subjects studied. The detectability of <i>RHD</i> sequence in the maternal plasma was determined by the same real-time PCR systems using 5 µl plasma DNA as templates. All experiments were carried out in duplicates. A sample would be scored as positive if any of the duplicates was positive. The presence of fetal DNA in the maternal plasma was confirmed by the amplification of <i>SRY</i> sequence (for male fetuses) and the <i>RASSF1A</i> sequence after enzymatic digestion from the maternal plasma DNA. The real-time PCR targeting the <i>SRY</i> gene was carried out using plasma DNA without enzymatic digestion as the templates. The real-time PCRs for <i>RASSF1A</i> and <i>β-actin</i> sequences were carried out using maternal plasma DNA after enzymatic digestion as the templates.

RESULTS

0089The RhD status of 355 pregnant women was screened. Fifty-four of them were <i>RHD</i> negative. As this group of subjects were at risk of alloimmunization by a RhD positive fetus, their plasma and CVS were subjected to further investigation for fetal RhD status. <i>RHD</i> sequences were detected in the maternal plasma DNA of 35 subjects and were negative in the maternal plasma of 19 subjects. In 15 of the 19 subjects with negative maternal plasma <i>RHD</i> result, <i>RASSF1A</i> sequences were detected in the plasma DNA after enzymatic digestion. The other 4 cases were negative for <i>RASSF1A</i> after enzymatic digestion. <i>Beta-actin</i> signal was negative in all cases indicating that the <i>BstU</i> I enzyme digestion was complete in all 19 cases. Based on the analysis of the CVS samples, all 15 subjects with positive detection of <i>RASSF1A</i> after enzymatic digestion were carrying a RhD-negative fetus. In the 4 subjects showing negative detection of <i>RHD</i> and <i>RASSF1A</i> in their plasma, the CVS were <i>RHD</i> positive in 2 of them. Thus, for these two cases, maternal plasma <i>RHD</i> genotyping had produced false negative results, which were picked up by the failure to detect the positive analytical marker <i>RASSF1A</i> after enzymatic digestion. To illustrate the importance of this gender independent fetal marker, these results were compared with an existing fetal DNA marker <i>SRY.</i> The <i>SRY</i> assay would be positive only when the fetus is male. For the 19 subjects with negative detection of <i>RHD</i> sequence in their plasma, 6 of them were positive for <i>SRY,</i> indicating the presence of amplifiable fetal DNA in the analyzed maternal plasma sample and thus further confirmed the genuine nature of the RhD-negative status of the fetus. In the remaining 13 cases, whether the negative detection of <i>RHD</i> and <i>SRY</i> sequences in the plasma DNA is a result of a female RhD negative fetus or the inadequate fetal DNA in the maternal plasma cannot be ascertained without using the <i>RASSF1A</i> protocol as a positive control for fetal DNA.

Example 6: Demonstration of <i>RASSF1A</i> as A Gender-Independent Marker for Monitoring Preeclampsia

0090The clinical utility of the hypermethylated fetal DNA markers goes beyond serving as a positive analytical marker. The detection and/or quantification of the <i>RASSF1A</i> by itself after enzymatic digestion can be useful in prenatal diagnosis and pregnancy monitoring. In other words, these hypermethylated fetal DNA sequences in maternal plasma can serve as biomarkers by their own right. Previously, it has been demonstrated that fetal DNA concentration in maternal plasma is increased in certain conditions such as preeclampsia and fetal aneuploidies. However, due to the lack of a gender-independent fetal DNA marker, previous studies were limited to pregnant women carrying a male fetus (<nplcit id="ncit0039" npl-type="s"><text>Levine et al., Am J Obstet Gynecol 190:707-713</text></nplcit>; <nplcit id="ncit0040" npl-type="s"><text>Leung et al., Clin Chem 47:147-139</text></nplcit>). In this example, we have compared the fetal DNA concentrations in the plasma, by targeting the <i>RASSF1A</i> sequence after enzymatic digestion, of 5 women suffering from preeclampsia with 5 gestational-age matched pregnant women without any pregnancy associated complication. The <i>RASSF1A</i> concentrations measured in the maternal plasma DNA samples after enzymatic digestion of the two groups are shown in <figref idref="f0022">Figure 16</figref>. The median concentrations of the pregnant women with and without preeclampsia were 9400 copies/ml and 2200 copies/ml plasma, respectively. The difference between the two groups was statistically significant (p=0.016, Mann-Whitney test).

0091All patents, patent applications, and other publications cited in this application, including published amino acid or polynucleotide sequences, are incorporated by reference in the entirety for all purposes. <tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1</b> Clearance of <i>RASSF1A</i> and <i>SRY</i> sequences from maternal plasma after delivery.</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="26mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><colspec colnum="4" colname="col4" colwidth="27mm" /><colspec colnum="5" colname="col5" colwidth="36mm" /><thead valign="top"><row><entry align="center" /><entry namest="col2" nameend="col5" align="center">After enzymatic digestion, plasma concentrations of (copies/ml)</entry></row><row><entry align="center" /><entry namest="col2" nameend="col3" align="center"><i>RASSF1A</i></entry><entry namest="col4" nameend="col5" align="center"><i>SRY</i></entry></row><row><entry align="center">Subjects</entry><entry align="center">Before delivery</entry><entry align="center">24 hours after delivery</entry><entry align="center">Before delivery</entry><entry align="center">24 hours after delivery</entry></row></thead><tbody><row><entry align="center">A</entry><entry align="center">84</entry><entry align="center">0</entry><entry align="center">27</entry><entry align="center">0</entry></row><row><entry align="center">B</entry><entry align="center">49</entry><entry align="center">0</entry><entry align="center">13</entry><entry align="center">0</entry></row><row><entry align="center">C</entry><entry align="center">42</entry><entry align="center">0</entry><entry align="center">15</entry><entry align="center">0</entry></row><row><entry align="center">D</entry><entry align="center">23</entry><entry align="center">0</entry><entry align="center">11</entry><entry align="center">0</entry></row><row><entry align="center">E</entry><entry align="center">19</entry><entry align="center">0</entry><entry align="center">9</entry><entry align="center">0</entry></row></tbody></tgroup></table></tables>

0092Blood was taken from 5 pregnant women carrying a male fetus just before delivery and 24 hours after delivery. After the maternal plasma DNA samples were treated by the methylation sensitive enzyme, <i>RASSF1A</i> and <i>SRY</i> sequences were detected in the plasma of all subjects before delivery, but were not detectable in any of the plasma samples at 24 hours after delivery. <tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2</b> The <i>RASSF1A</i> genotypes of the maternal buffy coat DNA, placental DNA, and maternal plasma DNA with or without enzyme digestion of 43 pregnant women. In each of the 43 cases, the genotype of the maternal plasma DNA with enzyme digestion was identical to the placental (fetal) genotype, suggesting that only fetal-specific DNA molecules were amplifiable after the enzyme digestion of the maternal plasma DNA samples.</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="26mm" /><colspec colnum="2" colname="col2" colwidth="32mm" /><colspec colnum="3" colname="col3" colwidth="41mm" /><colspec colnum="4" colname="col4" colwidth="32mm" /><colspec colnum="5" colname="col5" colwidth="36mm" /><thead valign="top"><row><entry align="center" /><entry namest="col2" nameend="col5" align="center"><b><i>RASSF1A</i> genotype</b></entry></row><row><entry align="center"><b><i>Case</i></b></entry><entry align="center"><b><i>Maternal buffy coat DNA</i></b></entry><entry align="center"><b><i>Maternal plasma DNA without Digestion</i></b></entry><entry align="center"><b><i>Placental DNA</i></b></entry><entry align="center"><b><i>Maternal plasma DNA with Digestion</i></b></entry></row></thead><tbody><row><entry align="center"><b>616</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>677</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>688</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>695</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>832</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>849</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>873</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>920</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>928</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1082</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1088</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1089</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1112</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1114</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1145</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1148</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1149</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1155</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1157</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1158</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1170</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1171</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1172</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1182</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1185</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1186</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1192</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1194</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1195</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1197</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1200</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1203</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry></row><row><entry align="center"><b>1204</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1210</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1211</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1212</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1213</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1214</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1222</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1234</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1235</b></entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry><entry align="center">CC</entry></row><row><entry align="center"><b>1266</b></entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry><entry align="center">AC</entry></row><row><entry align="center"><b>1276</b></entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry><entry align="center">AA</entry></row></tbody></tgroup></table></tables>