Patent Publication Number: US-2023151409-A1

Title: Methods and compositions for noninvasive prenatal diagnosis through targeted covalent labeling of genomic sites

Description:
TECHNICAL FIELD 
     This invention relates to the field of genetic testing for pregnant females in order to diagnose chromosomal aneuploidy and fetal gender from maternal peripheral blood samples. 
     BACKGROUND ART 
     Fetal chromosomal aneuploidy results from the presence of abnormal dose(s) of a chromosome or chromosomal region. The Down syndrome or Trisomy 21 (T21) is the most common incurable chromosomal aneuploidy in live born infants, which is typically associated with physical and mental disability (Parker et al. 2010). The overall incidence of T21 is approximately 1 in 700 births in the general obstetrical population, but this risk increases to 1 in 35 term births for women 45 years of age. An invasive diagnostic procedure is currently the only way to confirm the diagnosis of T21, commonly by a fetal cytogenetic analysis (such as karyotyping), which requires fetal genetic material to be invasively obtained by amniocentesis, chorionic villus sampling or cordocentesis. Due to the current risk of prenatal testing it is currently offered only for women in the high-risk group. Although the safety of invasive procedures has improved since their introduction, a well-recognized risk of fetal loss (0.5 to 1% for chorionic villus sampling and amniocentesis) and follow-up infections still remain (Akolekar et al. 2015). Hence, non-invasive and highly confident prenatal screening tests to reduce the number of invasive diagnostic procedures are still required. 
     Since the discovery of fetal genomic material in the form of circulating cell-free fetal DNA (cffDNA) in the blood plasma of pregnant women (Lo, et al., 1997) many attempts have been made aiming at using cffDNA for non-invasive risk-free prenatal testing (NIPT). Early applications of NIPT included the determination of Rhesus D blood-group status and fetal sex as well as the diagnosis of autosomal dominant disorders of paternal inheritance by quantitative real time PCR (qPCR) (Lo et al., 1998; Daniels et al, 2006). However, the application of cffDNA to the prenatal detection of fetal chromosomal aneuploidies has represented a considerable challenge. First of all, the cffDNA represents only a subfraction of 6-10% of the total cfDNA (cell-free DNA) of maternal origin in first and second trimester pregnancies and rises up to 10-20% in third trimester pregnancies (Lun et al., 2008; Lo et al., 2010), and this can often interfere with the analysis of fetal nucleic acids. One way to deal with the low abundance of the fetal DNA was the evaluation of the dosage of chromosome 21 calculating the ratios of polymorphic alleles in the placenta-derived DNA/RNA molecules (Lo, and Chiu, 2007). However, this method can only be applied to fetuses that are heterozygous for the targeted polymorphisms. 
     A study of Zimmermann et al (2002) was able to distinguish between trisomic 21 and euploid fetuses using qPCR based on the 1.5-fold increase in chromosome 21 dosage in the trisomic cases. Since a 2-fold difference in DNA template concentration constitutes a difference of only one threshold cycle (C T ), the discrimination of a 1.5-fold difference is at the limit of conventional qPCR. 
     With the development of massive parallel sequencing (MPS) the detection of fetal aneuploidy is carried out through counting cfDNA molecules and measuring the over- or underrepresentation of any chromosome in maternal plasma. As previous reports have indicated that fetal cffDNA is shorter than its maternal counterpart (Chan et al, 2004; Li et al, 2004; Fan et al, 2010), MPS has been combined with size fractionation prior to sequencing or in silico of plasma DNA fragments to enrich for fetal DNA. However, even though MPS has been widely used in commercial prenatal testing, such an approach which requires deep coverage or paired-end sequencing, increases the cost of service. 
     An alternative approach to improve the sensitivity and cost-effectiveness of NIPT is preferential targeting of fetal DNA sequences by utilizing epigenetic differences between maternal blood DNA and cffDNA. 
     Bisulfite conversion that enables analysis of the methylation status of each CG site, followed by either methylation-specific PCR or sequencing has been applied to detect methylation differences between maternal and fetal DNA (Chim, et al. 2005; Chiu, et al. 2007; Chim, et al. 2008; Lun et al, 2013; Jensen et al, 2015). However, although providing high resolution, bisulfite treatment reinforces the degradation of low amounts of fetal DNA, complicating fetal specific methylome analysis. Furthermore, screening genomes for diagnostic of DMRs by whole-genome bisulfite-sequencing is technologically demanding and extremely expensive leading to an unnecessary increase in cost of NIPT. 
     The application of methylation sensitive restriction digestion involves the use of methylation-sensitive restriction enzymes to remove hypomethylated maternal DNA thus allowing direct polymerase chain reaction (PCR) analysis of cffDNA (Old, et al. 2007; Tong et al, 2010). However, methylation sensitive restriction digestion is inherently limited by the sequence-specificity of available enzymes what restricts the number of DMR regions suitable for testing. 
     The methylcytosine-immunoprecipitation based approach (MeDIP) was used in combination with oligonucleotide array analysis, sequencing and MeDIP-qPCR for the quantification of selected hypermethylated fetal DMRs on chromosome 21 (Papageorgiou et al., 2009, Tsaliki et al, 2012, Keravnou et al, 2016). However, MeDIP enrichment is biased to highly methylated sequences (Weber et al. 2005) and thus, the potential diagnostic informativeness of the less CG dense or less methylated sequences might be lost. Therefore, further developments and advances are necessary for the identification and detection of highly specific and stable fetal-specific markers. 
     Placental DNA was reported to be generally hypomethylated as compared to maternal blood DNA. Examination of the differential methylation between placenta and maternal blood uncovered large contiguous genomic regions with significant placental hypomethylation relative to non-pregnant female cfDNA (Jensen et al, 2015). Moreover, these regions are of low CpG and gene density and thus could be poorly covered by affinity enrichment methods, such as MeDIP. Since unmodified CG fraction represents smaller portion of the human genome (20-30% of CGs are unmethylated), its targeted analysis is more relevant for cost-effective and sensitive detection of fetal specific DNA fragments in maternal circulation. 
     In recent years, we and others have been adapted covalent derivatization for epigenome-wide studies of various cytosine modifications (Song et al. 2011; Kriukienė et al. 2013; Staševskij et al. 2017; Gibas et al, 2020, accepted). Generally, robust and highly specific enrichment of a covalently modified minor fraction of cytosines in the fetal cffDNA, for example of unmodified CGs or hydroxymethylated cytosines, could potentially help achieve superior sensitivity and specificity in prenatal diagnostics. More importantly, a method for highly specific targeted analysis of a particular fraction of fetal regions combined with lower cost next generation sequencing devices or real time quantitative PCR (qPCR) can significantly alter the cost and turnaround time of NIPT, increasing the availability of NIPT screening for all pregnancies without the restriction to a high risk group. 
     SUMMARY OF INVENTION 
     In the first aspect, the present invention provides a new method for noninvasive prenatal diagnosis based on analysis of unmodified CG sites (uCG) or hydroxymethylated CGs (hmCGs) in nucleic acid molecules extracted from a biological sample obtained from a pregnant female typically during the first trimester of gestational age through use covalent modification of uCGs or hmCs and subsequent estimation of the labeled fraction of CG sites, enabling genome-wide identification of the fetal-specific regions. 
     According to one exemplary embodiment, a biological sample received from a pregnant female is analyzed to perform a prenatal diagnosis of a fetal chromosomal aneuploidy, such as trisomy T21, and fetal gender. 
     A maternal biological sample includes nucleic acid molecules found in various maternal body fluids, such as peripheral blood or a fractionated portion of peripheral blood, urine, plasma, serum, and other suitable biological samples. In a preferred embodiment, the maternal biological sample is a fractionated portion of maternal peripheral blood. 
     A large number of differentially labeled regions (DLRs) on chromosome 21, 13 and 18 which are differentially modified between non-pregnant female peripheral blood DNA sample and DNA of placental origin (chorionic villi (CV) of the fetal part of placenta which are enriched in fetal trophoblasts) or between non-pregnant female peripheral blood DNA sample and peripheral blood DNA sample of pregnant women have been identified using covalent chemical modification of the cytosine base of naturally unmodified CG sites or hydroxymethylated CG sites in maternal nucleic acid molecules. Subsequent PCR amplification with or without enrichment of the labeled fraction of CG sites coupled with sequence determination of the labeled and amplified nucleic acid molecules enabled genome-wide identification of the fetal-specific labeled regions. As used herein, the term DLR refers to a “differently labeled genomic region” that is more or less intensively labeled through enzymatic transfer of a reactive group onto the cytosine base in the nucleic acid molecule. For the purposes of the invention, the preferred DLRs (selected u-DLRs; see Table 4) are those that are hypomethylated and thus, more intensively labeled, in fetal DNA and hypermethylated in maternal DNA. In another aspect, the preferred DLRs (selected hm-DLRs; see Table 5) are those that are hyper-hydroxymethylated and thus, more intensively labeled, in fetal DNA and hypo-hydroxymethylated in maternal DNA. 
     In one embodiment, a DLR can be confined to a single cytosine or a dinucleotide, preferentially a CG dinucleotide (CG-DLRs). 
     Representative examples of a subset of these u-DLRs, hm-DLRs and CG-DLRs have been used to accurately predict trisomy 21, in a method based on analysis of fetal-specific hypomethylated or hydroxymethylated DNA in a sample of maternal blood, typically during the first trimester of gestational age. Thus, the effectiveness of the disclosed DLRs and methodologies for diagnosing fetal aneuploidies have been demonstrated. 
     In addition, representative examples of a subset of these u-DLRs and hm-DLRs have been used to accurately predict fetal gender from X and Y chromosomes, in a method based on analysis of fetal-specific hypomethylated DNA in a sample of maternal blood, typically during the first trimester of gestational age. Thus, the effectiveness of the disclosed DLRs and methodologies for diagnosing fetal gender have been demonstrated. 
     Accordingly, the invention pertains to a method for prenatal diagnosis of a trisomy 21, and fetal gender using a sample of maternal blood, the method comprising: 
     (a) enzymatic labeling of uCG and hmC sites of nucleic acid molecules in a sample of maternal blood with a first reactive group, preferably an azide group;
 
(b) chemically tethering of an oligodeoxyribonucleotide (ODN) having the second reactive group, preferably an alkyne group, to the first group in a template nucleic acid;
 
(c) producing nucleic acid molecules from a template nucleic acid sequence using a nucleic acid polymerase which contacts a template nucleic acid sequence at or around the site of the labeled uCG/hmC and starts polymerization from the 3′-end of a primer non-covalently attached to the ODN;
 
(d) determining the presence or availability of the CG target sites and hence the level of the unmodified or hydroxymethylated template genomic nucleic acid molecules across the regions of chromosomal DNA shown in Tables 4 or 5, or 6;
 
(e) comparing the acquired value of the regions of step (d) to a standard reference value for the combination of at least one region from the list shown in Tables 4-6, wherein the standard reference value is (i) a value for a DNA sample from a woman bearing a fetus without trisomy 21; or (ii) a value for a DNA sample from a woman bearing a fetus with trisomy 21.
 
(f) diagnosing a trisomy based on said comparison, wherein trisomy 21 is diagnosed if the acquired value of the regions of step (d) is (i) higher than the standard reference value from a woman bearing a fetus without trisomy 21; or (ii) lower than the standard reference value from a woman bearing a fetus without trisomy 21; or (iii) comparable to the standard reference value from a woman bearing a fetus with trisomy 21.
 
(g) detecting fetal gender based on said comparison wherein female gender of a fetus is detected if the acquired value of the regions of step (d) is comparable to the standard reference value from a woman bearing a female fetus, and male gender of a fetus is detected if the acquired value of the regions of step (d) is comparable to the standard reference value from a woman bearing a male fetus.
 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    is a diagram of the methodology for identification of Differentially Labeled Regions (DLRs) across chromosome 21 (or chromosomes 13 and 18) comparing the two tissue pairs: chorionic villi tissue DNA of the 1st trimester fetuses and fractionated peripheral blood DNA samples of non-pregnant controls and fractionated peripheral blood DNA samples of non-pregnant female and pregnant female carrying a healthy fetus from the 1st trimester pregnancies. Further strategy for area under curve (AUC) determination for diagnosing T21-affected fetuses is also shown. 
         FIG.  2    shows the difference in (a) uCG and (b) hmCG signal for the exemplary DLRs (tissue-specific u-DLR chr21:33840400-33840500; pregnancy-specific u-DLR chr21:33591700-33591800; tissue-specific hm-DLR chr21:35203200-35203300; pregnancy-specific hm-DLR chr21:43790900-43791000, selected from Tables 4 or 5) identified in chromosome 21 between chorionic villi tissue DNA of the 1st trimester fetuses and fractionated peripheral blood DNA samples of non-pregnant controls; and between fractionated peripheral blood DNA samples of non-pregnant female and pregnant female carrying a healthy fetus from the 1st trimester pregnancies (left panel). For diagnosing purposes of trisomy 21, the signal intensity across the exemplary DLRs is also shown for the samples of pregnant female carrying T21-diagnosed fetuses from the 1st trimester pregnancies (right panel). 
         FIG.  3    shows the difference in (a) uCG and (b) hmCG signal for the exemplary DLRs (u-DLR chr21:43933400-43933500; hm-DLR chr21:36053400-36053500; selected from the Tables 4 or 5) identified in chromosome 21 between fractionated peripheral blood DNA samples of pregnant female carrying a healthy fetus or a T21 diagnosed fetus from the 1st trimester pregnancies. 
         FIG.  4    shows the difference in mean signal of labeled individual CG-DLRs, namely, (a) u-CG-DLRs and (b) hm-CG-DLRs (selected from Table 6) in chromosome 21 for detection of fetal T21 aneuploidy. 
         FIG.  5    shows the difference in mean signal of labeled individual CG-DLRs, namely u-CG-DLRs (selected from Table 6) in chromosome X for fetal gender determination. Samples from pregnant women and fetal CV tissue were labeled either XX or XY according to the gender of a fetus, Female and Male, respectively. Samples from non-pregnant women, NPC, were labeled as None, 00. 
         FIG.  6    shows the relative quantification of individual or a combination of (a) u-CG-DLRs and (b) hm-CG-DLRs of fetal specific DNA regions located on chromosome 21 using real time quantitative PCR for replicated DNA samples of peripheral blood plasma DNA of women pregnant with healthy or T21-diagnosed fetuses. Y-axis indicates the threshold cycle values (C T ) calculated in qPCR for the regions selected from Table 6 whose genome coordinates are shown above the graphs. Notably, numerical values of C T  inversely correlate to the abundance of the DLR region, indicating higher abundance of the region in the blood samples of pregnant female carrying a T21-diagnosed fetus. 
         FIGS.  7   a  and  b    show simulation of a PCR-based test for fetal gender determination by measuring DNA methylation differences in (a) chromosome X or (b) chromosome Y, according to the scheme shown in  FIG.  8   c   . DNA of the 1st trimester CV tissue of both genders was mixed with nonpregnant female peripheral blood plasma DNA to the ratio 20/80 or 0/100, respectively, and the difference in the threshold cycle was evaluated by qPCR. ΔC T  indicates the difference in the threshold cycle values between the mixtures using the CV samples of both genders (indicated as XX and XY for female and male genders, respectively).  FIG.  7   c    shows relative quantification of fetal specific DNA regions located on chromosome X for fetal gender determination using qPCR for the replicated DNA samples of untreated, i.e. non-preamplified, pregnant female peripheral blood plasma, according to the scheme shown in  FIG.  8     c.    
         FIG.  8    is a schematic illustration of the analytical approach for calculation of DLRs using labeling and enrichment of unmodified CG or hydroxymethylated CG sites coupled with analysis by (a) real time quantitative PCR of pre-amplified samples; (b) sequencing of labeled CGs; (c) real time quantitative PCR of non-preamplified DNA samples, of fractionated peripheral blood DNA of pregnant female. ODN—the attached deoxyribonucleotide, A1/A2—the two strands of the ligated to DNA fragments partially complementary adaptors. 
         FIG.  9    shows the difference in (a) uCG and (b) hmCG signal for the exemplary DLRs (selected from Table 7; the genomic coordinates are shown above the graphs) identified for chromosome 13 and chromosome 18 between CV tissue DNA of the 1st trimester fetuses and fractionated peripheral blood DNA samples of non-pregnant controls; and between fractionated peripheral blood DNA samples of non-pregnant female and pregnant female carrying a healthy fetus from the 1st trimester pregnancies. 
         FIG.  10    shows the relative quantification of (a) u-CG-DLRs and (b) hm-CG-DLRs of T21 fetal-specific DNA regions located on chromosome 21 using real time quantitative PCR for an independent group of peripheral blood plasma DNA samples of women pregnant with healthy or T21-diagnosed fetuses. Y-axis indicates the threshold cycle values (C T ) calculated in qPCR for the regions selected from Table 6. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     In the present embodiment, the method comprises the measurement of the presence or availability of the target CG sites in the template nucleic acid molecules by sequencing of the amplified nucleic acid molecules of the biological sample, such that only the sequence of the targeted CGs and hence the unmodified/hydroxymethylated fraction of CGs is determined. In this embodiment, amplification prior to sequencing is performed through the ODN-directed and ligation-mediated PCR using one primer bound complementary to the ODN or a part of it in the absence of complementarity to the genomic template region, and the second primer bound through non-covalent complementary base pairing to oligonucleotide linkers ligated to both ends of the template nucleic acid molecule. In another aspect of this embodiment, amplification prior to sequencing can be performed by targeted PCR amplification utilizing one primer bound complementary to the ODN or a part of it in the presence (5-7 nucleotides complementarity to the genomic template DNA in the proximity of a CG site) or absence of complementarity to the genomic template DNA, and the second primer bound through non-covalent complementary base pairing to the template DNA in the chromosomal regions shown in Tables 4 or 5 or 6 or 7. 
     In further embodiments, the method comprises the measurement of the presence or availability of the labeled target sites and hence the level of the unmodified or hydroxymethylated template nucleic acid molecules by real time quantitative polymerase chain reaction (qPCR) of the enriched fetal CGs and DNA regions, which have been previously covalently targeted and pre-amplified using attached ODN as described above, utilizing one primer with its 5′ end bound complementary to the chromosomal regions shown in Tables 4-7 in the very close vicinity (its 5′ end binds at or more than 5 nucleotides to a labeled CG site) to the labeled cytosine, and the second primer bound complementary to the template DNA in the selected chromosomal regions shown in Tables 4 or 5 or 6 or 7. 
     In yet another aspect, the method comprises the measurement of the presence or availability of the labeled target sites and hence the level of the unmodified or hydroxymethylated template nucleic acid molecules in a non-preamplified DNA sample by real time quantitative polymerase chain reaction, utilizing one primer that recognizes and binds to the ODN and 5-7 nucleotides adjacent to the target CG site in a template genomic DNA through non-covalent complementary base pairing, and a second primer binds complementary to the template DNA in the selected chromosomal regions shown in Tables 4 or 5 or 6 or 7. 
     In the preferred embodiment of the invention, the plurality of differentially labeled regions (DLRs) preferably is chosen from the lists shown in Tables 4-7. In various embodiments, the levels of the plurality of DLRs are determined for at least one DLR, for example chosen from the lists shown in Tables 4-7. Preferably, the levels of the plurality of DLRs in the labeled DNA sample are determined by real time quantitative polymerase chain reaction (qPCR). As used herein, the term “a plurality of DLRs” is intended to mean one or more DLRs (or CG dinucleotides). 
     In a further aspect, the present invention pertains to a kit, comprising the composition of the invention. In other embodiments, the kit further comprises: 
     (a) an enzyme capable of covalent derivatization of the cytosine base with an active group, preferentially an azide group;
 
(b) a compound comprising the active group (an azide group);
 
(c) an ODN attached to the second reactive group, preferably an alkyne group; and
 
(d), oligonucleotide primers (e.g., two or more) for assessment of DLR regions through PCR amplification, wherein one primer binds to the ODN or in the close vicinity to the ODN attachment site through non-covalent complementary base pairing and is able to prime a nucleic acid polymerization reaction from the labeled CG and the second primer binds to the genomic regions described in Tables 4-7;
 
(e) in another embodiment, the kit can further comprise oligonucleotide linkers for ligation and/or oligonucleotide primers for PCR amplification of the nucleic acid molecules to be analyzed by qPCR or sequencing.
 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is based, at least in part, on the inventors&#39; identification of a large panel of differentially labeled regions (DLRs) and CGs (CG-DLRs) that exhibit strong labeling in fetal DNA and weak or absence of labeling in maternal DNA. Still further, the invention is based, at least in part, on the inventors&#39; demonstration that hypomethylated/hydroxymethylated fetal DNA can be specifically targeted and enriched through covalent modification of CGs, thereby resulting in a sample enriched for fetal DNA. Still further, the inventors have accurately diagnosed trisomy 21 and fetal gender in a panel of maternal peripheral blood samples using representative examples of the DLRs disclosed herein, thereby demonstrating the effectiveness of the identified DLRs and disclosed methodologies in diagnosing fetal aneuploidy T21 and fetal gender. 
     Various aspects of this disclosure are described in further detail in the following subsections. 
     I. A Method for Non-Invasive Detection of Fetal Aneuploidy T21 and Fetal Gender 
     Accordingly, the invention pertains to a method for prenatal diagnosis of a trisomy 21, and fetal gender using a sample of maternal blood, the method comprising: 
     (a) enzymatic labeling of uCG or hmC sites of nucleic acid molecules in a sample of maternal blood with a reactive azide group;
 
(b) chemically tethering of an oligodeoxyribonucleotide (ODN) having an alkyne group to the introduced azide groups in a template nucleic acid;
 
(c) producing nucleic acid molecules from a template nucleic acid sequence starting at the azide-labeled CG sites through PCR amplification;
 
(d) determining the labeling intensity level of unmodified or hydroxymethylated template genomic nucleic acid molecules across the regions or CG sites of chromosomal DNA shown in Tables 4 or 5, or 6 using, preferably qPCR, or sequencing of labeled genomic fraction;
 
(e) comparing the experimentally acquired value of the regions of step (d) to a standard reference value for the combination of at least one region, or at least two regions from the list shown in Tables 4-6, wherein the standard reference value is (i) a value for a DNA sample from a woman bearing a fetus without trisomy 21; or (ii) a value for a DNA sample from a woman bearing a fetus with trisomy 21.
 
(f) diagnosing a trisomy 21 based on said comparison, wherein trisomy 21 is diagnosed if the experimentally acquired value of the sample is (i) higher than the standard reference value from a woman bearing a fetus without trisomy 21; or (ii) lower than the standard reference value from a woman bearing a fetus without trisomy 21; or (iii) comparable to the standard reference value from a woman bearing a fetus with trisomy 21.
 
     A schematic illustration of the analytical approach for evaluation of labeling intensity in DLRs using labeling and enrichment of unmodified or hydroxymethylated CGs is demonstrated in  FIG.  8   . 
     II. Labeling of Unmodified or Hydroxymethylated CG Sites 
     Methods for the first step of covalent derivatization of genomic DNA sites are known in the art. Covalent labeling of genomic uCG or hmC sites can be performed using an enzyme capable of transfer of a covalent group onto genomic DNA. The enzyme may comprise a methyltransferase or a glucosyltransferase. 
     An enzyme for covalent labeling of uCG sites is preferably the C5 DNA methyltransferase M.Sssl or a modified variant of it, such as M.Sssl variant Q142A/N370A (Kriukiene et al., 2013; Stasevskij et al, 2017) which is adapted to work with synthetic cofactors, such as Ado-6-azide cofactor (Kriukiene et al., 2013; Masevicius et al., 2016). 
     An enzyme for covalent labeling of hmC/hmCG sites is preferably the phage T4 beta-glucosyltransferase (BGT) which is adapted to work with synthetic cofactors, such as UDP-6-azidoglucose (Song et al, 2011). 
     The ODN is preferably from 20 to 90 nucleotides in length, as shown in the exemplary embodiment preferably 39 nt. The ODN contains the reactive group at the second base position from its 5′-end, preferably the alkyne group, which reacts with the azide group which was enzymatically introduced in a template nucleic acid molecule. 
     It should be noted that DNA after covalent labeling becomes enzymatically and chemically altered but preserves base specificity. As used herein, the term “enzymatically altered” is intended to mean reacting the DNA with an enzymatically transferred chemical group that enables the conversion of respective CG sites into the azide-CG sites, giving discrimination of the labeled sites from template CGs. As used herein, the term “chemically altered” is intended to mean enzymatic transformation of template cytosine into the azide-modified cytosine in CG sites. Thus, in the instant method the fetal specific regions are calculated between more intensively and less intensively labeled CG sites in DNA without the need to directly determine methylation or hydroxymethylation levels of template DNA. Furthermore, in the instant method, the DNA of the maternal blood sample is not subjected to sodium bisulfite conversion or any other similar chemical reactions that alter base specificity, such as sodium bisulfite conversion, nor the maternal blood sample is treated with a methylation-sensitive restriction enzyme(s) or through direct or indirect immunoprecipitation to enrich for a portion of maternal blood sample DNA. 
     Alternatively, the ODN-derivatized template DNA can be enriched on solid surfaces using an affinity tag that is introduced in the composition of the ODN. A useful affinity tag preferably is but not restricted to the biotin and can be used in the methods of the present invention. In this aspect, the invention includes an additional step of separating maternal nucleic acid sequences on a solid surface, for example on streptavidin/avidin beads, thereby further enriching for nucleic acid molecules containing labeled CG sites. Other approaches known in the art for physical separation of components can be also used. The captured DNA is to be used for further analysis without detachment or can be detached from beads in mild conditions, such as, for example pure water and heating to 95° C. for 5 min. 
     III. Producing of Template Nucleic Acid Molecules from the Site of Covalent Labeling 
     In the diagnostic method, a nucleic acid polymerase primes polymerization of the template nucleic acid at or around the site of labeling using the 3′-end of an externally added primer which is non-covalently attached to the ODN. Non-covalent bonding preferably involves base pairing interaction between the ODN and the externally added primer. In the preferred embodiments shown in  FIGS.  8   a  and  b   , the structure of the ODN permits correct positioning of the externally added primer to the template at the site of the ODN attachment; the primer should be complementary to the sequence of the ODN while should not make any complimentary base pairing with the template nucleic acid at its 3′-end. In yet another aspect, shown in  FIG.  8   c    the primer at its 5′-end should be complementary to the sequence of the ODN while its 3′-end should make complementary base pairing with preferably at least 5 nucleotides and not more than 7 nucleotides of the template nucleic acid that are adjacent to the site of the attached ODN. 
     In the diagnostic method, typically after tagging of CGs in the maternal blood sample with the ODN, the tagged CGs and adjacent template nucleic acid are pre-amplified starting from the site of the attachment of the ODN. As used herein, the term “pre-amplified” is intended to mean that additional copies of the DNA are made to thereby increase the number of copies of the DNA, which is typically accomplished using the polymerase chain reaction (PCR). 
     In the preferred embodiment, the experimentally acquired value for the presence or availability of labeled CG that were tagged with the ODN in the maternal blood sample can be acquired by amplification of the DNA molecules starting from the tagged CG sites using the ODN-directed and partially ligation mediated (LM-PCR) polymerase chain reaction. The skilled person will be well aware of suitable methods for ligating adaptor sequences to the DNA fragments. In LM-PCR of the present invention, an adaptor nucleic acid sequences are added onto both ends of each DNA fragments through preferably sticky end or blunt-end ligation, wherein each strand of an adaptor sequences is capable of hybridizing with a primer for PCR, thereby amplifying the DNA fragments to which the linkers have been ligated. In this aspect of the present invention, only one strand of the ligated partially complementary double-stranded adaptor sequence is used to anchor a primer for amplification of the labeled template DNA strand as shown in  FIG.  8   b   . The second primer binds to the ODN sequence through complementary base pairing without contacts to the template DNA. The externally added primer should be at least 10 nucleotides and preferably at least 15 nucleotides in order to allow for a section of a primer to be involved in base pairing with the ODN without the complementary base pairing with the template DNA. This results in amplification of the labeled strands of nucleic acid samples, but not the original DNA fragment to which the adaptor sequences were ligated. In a preferred embodiment, the values of the amplified sequences are determined through real time quantitative polymerase chain reaction using oligonucleotide primers annealing within the regions shown in Tables 4, 5, 6 or 7 in the close vicinity to the labeled CGs as shown in  FIG.  8   a   . Methods of qPCR are well known in the art. Representative, non-limiting conditions for qPCR are given in the Examples. 
     Yet, alternatively, the values of the amplified sequences, or DLRs, are determined through massive parallel sequencing. In this aspect of the embodiments, one strand of the ligated double-stranded adaptor sequence is used to anchor a primer for amplification of the labeled template DNA strand as shown in  FIG.  8   b   . The second primer binds to the ODN sequence through complementary base pairing without contacts to the template DNA. Following PCR amplification, the values of the amplified sequences are determined through sequencing. This is only one exemplification of the presently described strategy for estimation of labeled nucleic acid through sequencing. In yet another aspect, the sub-fraction of the derivatized maternal sample DNA is selectively enriched through targeted PCR amplification prior to sequencing. Such PCR amplification makes use one primer bound complementary to the ODN or a part of it in the presence (5-7 nucleotide complementarity right at the target sites) or absence of complementarity to the template DNA, and the second primer bound through non-covalent complementary base pairing to the template DNA in the chromosomal regions shown in Tables 4-7. 
     In another embodiment of the invention, the experimentally acquired value for the presence or availability of labeled CG is estimated through qPCR, in a maternal blood sample that has not been subjected to adaptor ligation or pre-amplification, as shown in  FIG.  8   c   . In this aspect, one primer to be used in qPCR hybridizes complementarily to the ODN altogether with 5-7 nucleotides of genomic template DNA near the derivatized CG site as described above and the second primer binds within the genomic DNA positions listed in Tables 4-7. 
     IV. Differentially Labeled Regions (DLRs). 
     The diagnostic method of the invention employs a plurality of regions of chromosomal DNA wherein the regions are more intensively labeled in fetal DNA as compared to female peripheral blood samples. In theory, any chromosomic region with the above characteristics can be used in the instant diagnostic method. In particular, methods for identifying such DLRs are described in detail below and in the Examples (see Examples 1 and 2). Moreover, a large panel of DLRs for chromosomes 21, 13 and 18 suitable for use in the diagnostic methods, has now been identified (the strategy for identification of DLRs is shown in  FIG.  1   ). 
     Furthermore, representative examples of a subset of these DLRs (4175 tissue-specific u-DLRs; 163 pregnancy-specific u-DLRs; 8815 tissue-specific hm-DLRs, 679 pregnancy-specific hm-DLRs) have been used to accurately predict trisomy 21, in a method based on analysis of fetal-specific DLRs in chromosome 21 by sequencing of labeled CG sites in a maternal blood sample. We also evaluated labeling differences between maternal blood samples of healthy and T21 positive pregnancies and identified 3,490 u-DLRs and 2,002 hm-DLRs which are shown in Tables 4 and 5, respectively. The effectiveness of the disclosed regions and methodologies for diagnosing fetal aneuploidy T21 has been demonstrated in  FIGS.  2  and  3   . Such DLRs are shown in the lists of Tables 4 and 5, which provide the selected DLRs for chromosome 21. 
     According to the second exemplary embodiment, DLRs restricted to individual CGs (CG-DLRs) have been identified in chromosomes 21 and X. Representative examples of a subset of these DLRs have been used to accurately predict trisomy 21, in a method based on analysis of fetal-specific hypomethylated or hyper-hydroxymethylated CG-DLRs in chromosome 21 by sequencing of labeled CG sites in a sample of maternal blood. Also, representative examples of a subset of these CG-DLRs have been used to accurately predict fetal gender, in a method based on analysis of fetal-specific CG-DLRs in chromosome X by sequencing of labeled CG sites in a sample of maternal blood. The effectiveness of the disclosed DLRs and methodologies for determination T21 aneuploidy and fetal gender has been demonstrated in  FIG.  4    and  FIG.  5   . The list of DLRs is shown in Table 6. 
     In the third exemplary embodiment, representative examples of a subset of the CG-DLRs have been used to accurately predict trisomy 21 and fetal gender, in a method based on analysis of fetal-specific DLRs in chromosome 21 and chromosome X and/or Y in a sample of maternal blood by qPCR. Thus, the effectiveness of the disclosed regions and methodologies for diagnosing trisomy 21 and fetal gender has been demonstrated in  FIG.  6    and  FIG.  7   . 
     In other methods for detecting a fetal aneuploidy, the plurality of DLRs may be on chromosome 13, chromosome 18, to allow for diagnosis of aneuploidies of any of these chromosomes. In theory, any DMR with the above characteristics in a chromosome of interest can be used in the instant diagnostic method. Methods for identifying such DLRs in chromosome 13 and chromosome 18 are described in Example 1 and the effectiveness of the disclosed regions has been demonstrated in  FIG.  9   . The lists of selected DLRs for chromosomes 13 and 18 are provided in Table 7. 
     As used herein, the term “a plurality of DLRs” is intended to mean one or more regions or DLRs, selected from the list shown in Table 4-7. In various embodiments, the levels of the plurality of DLRs are determined for at least one region. Control regions or control DLRs also can be used in the diagnostic methods of the invention as a reference for evaluation of the labeled signal in the DLR region(s) of interest. 
     In a particularly preferred embodiment, the plurality of DLRs on chromosome 21 comprise one region or a combination of at least two regions, selected from the group shown in Table 6. 
     The invention also pertains to a composition comprising nucleic acid probes that selectively detect DLRs shown in Table 6. 
     The actual nucleotide sequence of any of the DLRs shown in Tables 4-7 is obtainable from the information provided herein together with other information known in the art. More specifically, each of the DLRs shown in Tables 4-7 is defined by a start base position on a particular chromosome, such as, for example “position 10774500” of chromosome 21. Furthermore, primers for targeted detection and/or amplification of a DLR can then be designed, using standard molecular biology methods, based on the nucleotide sequence of the DLR. 
     In another aspect, the invention provides nucleic acid compositions that can be used in the methods and kits of the invention. These nucleic acid compositions are informative for detecting DLRs. As described in detail in Example 3, at least one CG-DLR shown in Table 6 has been selected and identified as being sufficient to accurately diagnose trisomy 21 in a maternal blood sample during pregnancy of a woman bearing a trisomy 21 fetus. 
     V. Determining Levels of DLRs. 
     Labeling levels of the identified DLRs can be measured by sequencing or by qPCR. 
     Labeling levels of a plurality of regions as described above are determined in the unmethylated or hydroxymethylated DNA sample, to thereby obtain a labeling value for the DNA sample. As used herein, the term “the levels of the plurality of DLRs are determined” is intended to mean that the prevalence of the DLRs is determined. The basis for this is that in a fetus with a fetal trisomy 21 there will be a larger amount of the DLRs as a result of the trisomy 21, as compared to a normal fetus. In another aspect, when the T21-specific DLR are being used, the amount of such DLRs can be larger or lesser then the amount in a fetus without a fetal trisomy 21. 
     In a preferred embodiment, the levels of the plurality of DLRs are determined by real time quantitative polymerase chain reaction (qPCR), a technique well-established in the art. The term “the labeling value” is intended to encompass any quantitative representation of the level of DLRs in the sample. For example, the data obtained from qPCR can be used as “the labeling value” or it can be normalized based on various controls and statistical analyses to obtain one or more numerical values that represent the level of each of the plurality of DLRs in the testing DNA sample. The procedure for detection of DLRs by qPCR including primers&#39; sequences, and the cycle conditions used were as described in Example 3. 
     In analysis of labeling intensity of DLRs by sequencing, the level of differential labeling was calculated for non-overlapping 100 bp regions. In more detail, for each window we computed the total log-transformed coverage and the fraction of identified CGs which we then normalized by the total log-transformed coverage and the fraction of identified CGs in reference chromosomes 16 (for uCG signal) and 20 (for hmC signal). For each window a full and null logistic regression models were fitted. Full model included coverage, identified fraction, and, for T21-specific DMRs, fetal sex and fetal fraction, as independent variables. Coverage and identified fraction were excluded from the null model. ANOVA Chi-squared test was used to compare full and null models to obtain p value. In cases where models did not converge fetal sex was removed and p value evaluated again. Model statistics were moderated using empirical Bayes. FDR was used to adjust p values for multiple testing and q&lt;0.05 was used as significance threshold. 
     For each pregnancy-specific or tissue-specific DLR a leave-one-out cross-validation procedure was performed in order to determine its ability to diagnose T21. For each cross-validation cycle Bayesian generalized linear model (Gelman et al. 2008) with normalized coverage and identified CG as independent variables was constructed on the training samples. The model was then applied on the testing sample returning the predicted probability of the sample belonging to the T21 category. After all the cross-validation cycles the prediction probabilities for all samples were taken together. Various thresholds that would determine the discrete sample class from continuous probability measurement may have different effects on predictor&#39;s specificity and sensitivity. Therefore, a receiver-operating characteristic curve analysis was performed to estimate the effect of any threshold. The area under receiver-operating characteristic curve (AUC) indicates the overall accuracy of the model. Those DLRs for which the area under the curve was equal to 100% and, therefore, could achieve 100% prediction accuracy, were deemed to be the T21-predictive DLRs. 
     An approach that would combine individual DLRs into a single predictive model is also possible. Such model could be one of but not limited to elastic net, random forest or support vector machine. Model would be evaluated in the same way by assessing receiver-operating characteristic and using cross-validation for parameter tuning. Also, bootstrap could be used instead of cross-validation. Other model accuracy measures could be employed, and data could be transformed in different ways. Interactions of DLRs could be taken into account to build new composite features that would be used for subsequent model training and evaluation. 
     VI. Comparison to a Standardized Reference Value. 
     The labeling value of the fetal DNA (also referred to herein as the “test value”) present in the maternal peripheral blood is compared to a standardized reference value, and the diagnosis of trisomy 21 (or lack of such fetal trisomy 21) is made based on this comparison. Typically, the test value for the fetal DNA sample is compared to a standardized normal reference value for a normal fetus, and diagnosis of fetal trisomy 21 is made when the test value is higher than the standardized normal reference labeling value for a normal fetus. In another aspect, the test value can be lower than the standardized normal reference labeling value for a normal fetus. 
     Alternatively, the test value for the labeled DNA sample can be compared to a standardized reference labeling value for a fetal trisomy 21 fetus, and diagnosis of fetal trisomy 21 can be made when the test value is comparable to the standardized reference labeling value for a fetal trisomy 21 fetus. 
     To establish the standardized normal reference labeling values for a normal fetus, maternal blood samples from the pregnant women carrying a normal fetus are subjected to the same steps of the diagnostic method, namely amplification of the ODN-derivatized CGs and their neighboring genomic sequences to obtain a reference DNA sample, and then determining the labeling value and the levels of at least one region of chromosomal DNA by sequencing or qPCR wherein selected from Tables 4-7. 
     In order to establish the standardized normal reference methylation values for a normal fetus, healthy pregnant women carrying healthy fetuses or healthy non-pregnant women are selected. Pregnant women are of similar gestational age, which is within the appropriate time period of pregnancy for screening fetal chromosomal aneuploidy, typically within the first trimester of pregnancy. Standardized reference labeling values for a T21 fetus can be established using the same approach as described above for establishing the standardized reference values for a healthy fetus, except that the maternal blood samples used to establish the T21-specific reference values are from pregnant women who have been determined to be carrying a fetus with fetal trisomy 21. 
     EXAMPLES 
     Example 1. Identification of DLRs 
     This example provides the methodology for the preparation of the labeled genomic libraries of the mentioned-above biological samples for genomic mapping of unmodified or hydroxymethylated CGs. Also, this example provides the strategy for DLRs determination and how DLRs for detection of trisomy T21 were preferentially chosen.  FIG.  8   b    shows the application of the sequencing methodology for the identification of DLRs. In this example, DLRs in chromosomes 13 and 18 were also identified. 
     Biological Samples. 
     We performed analysis of three distinct sample types, enabling a characterization of the unmethylated and hydroxymethylated CGs in DNA obtained from plasma of pregnant women; we created single CG resolution uCG and 5hmCG maps of placental chorionic villi (CV) tissue samples from the 1st trimester abortions (CVS; n=6 of uCG and n=3 of 5hmCG); cfDNA samples of female non-pregnant controls (NPC; uCG n=6 and 5hmCG n=7) and cfDNA samples of pregnant women carrying healthy fetuses (uCG n=7 and 5hmCG n=6) or fetuses with the trisomy 21 (uCG n=5 and 5hmCG n=4). 
     Circulating DNA from maternal blood samples was extracted using the MagMax Nucleic Acid Extraction kit (Thermo Fisher Scientific (TS)) or the QIAamp DNA blood Midi Kit (QIAGEN), and DNA from chorionic villi tissue was prepared by phenol extraction. 
     All the maternal peripheral blood DNA samples (1st trimester pregnancies) and chorionic villi samples (1st trimester abortions) were obtained at Tartu University Hospital (Tartu, Estonia) through collaboration with Tartu University (Estonia). Consent forms approved by the Research Ethics Committee of the University of Tartu (ethical permission No. 246/T-21 and 213/T-21) were collected for each of the mother participated. 
     Mapping of Unmodified/Hydroxymethylated CGs in DNA Extracted from Biological Samples. 
     In uTOP-seq, 4-10 ng of cfDNA (or 100 ng of CV tissue DNA, sheared to 200 bp by Covaris sonicator) were labeled with 0.11 ΣM eM.Sssl (Kriukienė et al. 2013) in 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5 mM EDTA buffer supplemented with 200 μM Ado-6-azide cofactor (Masevicius et al, 2016) for 1 h at 30° C. followed by thermal inactivation at 65° C. for 20 min and Proteinase K treatment (0.2 mg/ml) for 30 min at 55° C. and finally column purified (GeneJET PCR purification kit, (TS)). In hmTOP-seq, 5hmC glycosylation was carried with 5-10 ng of cfDNA supplemented with 50 μM UDP-6-azide-glucose (Jena Bioscience) and 2.5-5 U T4 β-glucosyltransferase (TS) for 1 h 37° C. followed by enzyme inactivation at 65° C. for 20 min and column purification (GeneJET PCR Purification kit (TS)). After ligation of the partially complementary adapters as described previously (Staševskij et al. 2017), covalently labeled DNA was supplemented with 20 μM alkyne-containing DNA oligonucleotide (which was biotinylated for construction of 5hmC maps) (ODN; 5′-T(alkyneT)TTTTGTGTGGTTTGGAGACTGACTACCAGATGTAACA-3′ (or -(biotin)-3′), Base-click) and 8 mM CuBr: 24 mM THPTA mixture (Sigma) in 50% of DMSO, incubated for 20 min at 45° C. and subsequently diluted to &lt;1.5% DMSO before a column purification (GeneJET NGS Cleanup Kit, Protocol A (TS)). DNA recovered after biotinylation step was incubated with 0.1 mg Dynabeads MyOne Cl Streptavidin (TS) in a buffer A (10 mM Tris-HCl (pH 8.5), 1 M NaCl) at room temperature for 3 h on a roller. DNA-bound beads were washed 2× with buffer B (10 mM Tris-HCl (pH 8.5), 3 M NaCl, 0.05% Tween 20); 2× with buffer A (supplemented with 0.05% Tween 20); 1× with 100 mM NaCl and finally resuspended in water and heated for 5 min at 95° C. to recover enriched DNA fraction. Purified DNA after oligonucleotide conjugation (uCG) or biotin-enrichment (5hmC) was subsequently used in a priming reaction with 1 U Pfu DNA polymerase (TS), 0.2 mM dNTP, 0.5 μM complementary priming oligonucleotide (EP; 5′-TGTTACATCTGGTAGTCAGTCTCCAAACCACACAA-3). The reaction mixture was incubated at the following cycling conditions: 95° C. 2 min; 5 cycles at 95° C. 1 min, 65° C. 10 min, 72° C. 10 min. Amplification of a primed DNA library was carried out by adding the above reaction mixture to 100 μl of amplification reaction containing 50 μl of 2× Platinum SuperFi PCR Master Mix (TS) and barcoded fusion PCR primers A(Ad)-EP-barcode-primer (63 nt) and trP1(Ad)-A2-primer (45 nt) at 0.5 μM each. Thermocycler conditions: 94° C. 4 min; 15 cycles (uCG) or 17 cycles (5hmC) at 95° C. 1 min, 60° C. 1 min, 72° C. 1 min. The final libraries were size-selected for −270 bp fragments (MagJET NGS Cleanup and Size Selection Kit, (TS)), and their quality and quantity were tested on 2100 Bioanalyzer (Agilent). Libraries were subjected to Ion Proton (TS) sequencing. 
     Data Analysis. 
     Raw TOP-seq and hmTOP-seq sequencing reads were processed as described in Staševskij et al. (2017) and Gibas et al. (2020, accepted) except for the 3′ sequence ends where adapter sequences were trimmed only if they were identified using cutadapt with maximum allowed error rate 0.1 (Martin 2011). Processed reads were mapped to reference human genome version hg19 and coverage for each CG dinucleotide was computed as the total number of reads starting at or around the CG dinucleotide on either of its strands. We define CG coverage as the total number of reads, c, on any strand starting within absolute distance, d. We retained only reads with d≤3. Only reads aligned to chromosomes 1 to 22, X and Y were used for further analysis. On average, 40% of the raw reads were retained for downstream analysis per sample. 
     Outlier identification was performed separately for uCG and 5hmC samples. CG coverage matrices were transformed using Hellinger transformation (Legendre and Gallagher, 2001) and then represented in two-dimensional space using non-metric multidimensional scaling (nMDS) with Bray-Curtis similarity index (Bray and Curtis, 1957). Samples that were further than two standard deviations away from the mean of their own sample group (cfDNA of non-pregnant controls, other cfDNA, CV tissue) in either nMDS1 or nMDS2 dimension were deemed outliers and removed from further analysis. There were three outlying samples in uCG and one in 5hmCG dataset. 
     Identification of DLRs in Chromosomes 21, 13 and 18. 
     The strategy for DLR identification is show in  FIG.  1   . We partitioned the chromosome 21 or 13 or 18 into 100 bp-wide non-overlapping windows. For each window we computed the total log-transformed coverage and the fraction of CGs covered which we then normalized by the total log-transformed coverage and the fraction of identified CGs in reference chromosomes 16 (for uCG) and 20 (for hmC). 
     First, we obtained the pregnancy-specific u-DLRs by comparing NPC samples with cfDNA samples of healthy pregnancies. For each window a full and null logistic regression models were fitted. Full model included coverage, identified fraction, and, for T21-specific DLRs, fetal sex and fetal fraction, as independent variables. Coverage and identified fraction were excluded from the null model. ANOVA Chi-squared test was used to compare full and null models to obtain p value. In cases where models did not converge fetal sex was removed and p value evaluated again. Model statistics were moderated using empirical Bayes adjustment. FDR was used to adjust p values for multiple testing and q&lt;0.05 was used as significance threshold. 
     Next, we used the same strategy to obtain tissue-specific u-DLRs (FDR q&lt;0.05; logistic regression) by comparing NPC and CV tissue samples. The same analytic approach was used separately for uCG and hmCG data. In case of hm-DLRs, nominal p value threshold was used when analysis did not yield any FDR significant DLRs. 
     Further, for each hypomodified pregnancy-specific and tissue-specific u-DLR or hyper-hydroxymethylated pregnancy-specific and tissue-specific hm-DLR in chromosome 21 a leave-one-out cross-validation procedure was performed in order to determine its ability to diagnose T21. For each cross-validation cycle Bayesian generalized linear model (Gelman et al. 2008) with normalized coverage and identified CG as independent variables was constructed on the training samples. The model was then applied on the testing sample returning the predicted probability of the sample belonging to the T21 category. After all the cross-validation cycles the prediction probabilities for all samples were taken together. Various thresholds that would determine the discrete sample class from continuous probability measurement may have different effects on predictor&#39;s specificity and sensitivity. Therefore, a receiver-operating characteristic curve analysis was performed to estimate the effect of any threshold. The area under receiver-operating characteristic curve indicates the overall accuracy of the model. Those DLRs for which area under the curve was equal to 100% and, therefore, could achieve 100% prediction accuracy, were deemed to be T21-predictive DLRs ( FIG.  1   ). 
     Using the strategy for DLR determination in chromosome 21, we obtained 2,761 pregnancy-specific u-DLRs (FDR q&lt;0.05) and 16,555 fetal tissue-specific u-DLRs (FDR q&lt;0.05; logistic regression). For hm-DLR identification, we used nominal p&lt;0.05 threshold and identified 4,930 pregnancy-specific hm-DLRs and 15,986 tissue-specific hm-DLRs. 
     An in-depth investigation of the identified DLRs between non-pregnant female peripheral blood and placental DNA samples or non-pregnant and pregnant female cfDNA samples, has led to the selection of a list of DLRs located on chromosome 21 for diagnosing trisomy 21. The selection criteria of the regions were based firstly on the labeling intensity status of the regions in maternal blood samples and CV DNA samples, or on the labeling intensity status of the regions in the non-pregnant and pregnant female maternal blood samples. More specifically, the selected regions should demonstrate a high labeling intensity status in CV tissue DNA and a low labeling intensity or absence of labeling in peripheral blood samples of NPCs, or should show a high labeling intensity status in pregnant female blood samples and a low labeling intensity or absence of labeling in NPCs. Using leave-one-out cross-validation as described above we discovered 4175 tissue-specific u-DLRs; 163 pregnancy-specific u-DLRs; 8815 tissue-specific hm-DLRs, 679 pregnancy-specific hm-DLRs in chromosome 21 that classified the samples according to fetal karyotype with 100% accuracy (the selected DLRs are shown in Tables 4 and 5, for the uCG and hmCG signal, respectively) ( FIG.  2   ). 
     Furthermore, considering global epigenetic changes in Down syndrome affected fetuses (Jin et al. 2013), we also employed an alternative approach to identify the trisomy 21-specific DLRs. We evaluated modification differences between cfDNA samples of healthy and T21-diagnosed pregnancies and identified differentially modified DLRs. A logistic regression model was fitted to each 100 bp window with the CG-coverage and CG-fraction as independent variables and karyotype as the response variable, as above. In addition, we adjusted for possible confounding effects of fetal fraction and fetal gender which could not be accounted for in the previous analyses. With such approach, we identified 3,490 u-DLRs and 2,002 hm-DLRs (FDR q&lt;0.05; logistic regression). The selected T21-specific DLRs that discriminate most the sample groups of healthy and T21-diagnosed pregnancies are shown in Tables 4 and 5, for uCG and hmCG signal, respectively) ( FIG.  3   ). 
     Using the same strategy for DLR identification shown in  FIG.  1    we also identified DLRs in chromosomes 13 and 18. For chromosome 13, we obtained 1,394 pregnancy-specific u-DLRs (FDR q&lt;0.05) and 25,091 fetal tissue-specific u-DLRs (FDR q&lt;0.05; logistic regression) and using nominal p&lt;0.05 threshold 4,255 pregnancy-specific hm-DLRs and 22,526 tissue-specific hm-DLRs. For chromosome 18, we obtained 1,321 pregnancy-specific u-DLRs (FDR q&lt;0.05), 22,121 fetal tissue-specific u-DLRs (FDR q&lt;0.05; logistic regression) and 3,626 pregnancy-specific hm-DLRs and 20,780 tissue-specific hm-DLRs. The lists of the selected DLRs across chromosomes 13 and 18 are shown in Table 7 ( FIG.  9   ). 
     The total number of fetal specific hypomethylated and hyper-hydroxymethylated tissue- and pregnancy-specific DLRs identified across chromosomes 21, 13 and 18 is summarized in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Numbers of pregnancy- and tissue-specific DLRs  
               
               
                 identified across chromosomes 21, 13 and 18. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 No. of  
                 No. of  
                 No. of hyper- 
                 No. of hyper- 
               
               
                   
                 hypo- 
                 hypo- 
                 hydroxy- 
                 hydroxy- 
               
               
                   
                 methylated  
                 methylated 
                 methylated 
                 methylated 
               
               
                   
                 tissue- 
                 pregnancy- 
                 tissue- 
                 pregnancy- 
               
               
                   
                 specific  
                 specific  
                 specific 
                 specific 
               
               
                 Chromosome 
                 u-DLRs 
                 u-DLRs 
                 hm-DLRs 
                 hm-DLRs 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Chr21 
                 4175 
                 163 
                 8815 
                 679 
               
               
                 Chr13 
                 25091 
                 1394 
                 22526 
                 4255 
               
               
                 Chr18 
                 22121 
                 1321 
                 20780 
                 3626 
               
               
                   
               
            
           
         
       
     
     Example 2. Identification of Individual Labeled CGs for Detection of Trisomy 21 and Fetal Sex 
     This example provides the strategy for determination of individual labeled CGs (CG-DLRs) following analysis of the samples described in Example 1 that can be used for detection of fetal trisomy T21. 
     An investigation of labeling intensities of uCGs and hmCGs in peripheral blood samples of women that were confirmed to be carrying a fetus with trisomy 21 against labeling intensities of uCGs and hmCGs in the three types of control samples, i.e. placental CV tissue DNA, peripheral blood samples of non-pregnant women and peripheral blood samples of women pregnant with healthy fetuses, has led to the selection of individual CG-DLRs located on chromosome 21 for detection of fetal T21. The selection criteria of the CG-DLRs were based firstly on a labeling intensity status of CGs in blood samples of women pregnant with T21-diagnosed fetuses. More specifically, the selected CG-DLRs should demonstrate a high labeling intensity status in blood samples of women pregnant with T21-diagnosed fetuses and a low labeling intensity or absence of labeling in the three other sample types: CV tissue DNA, peripheral blood samples of NPC and pregnant female carrying a healthy fetus. 
     The CGs with non-zero coverage and non-zero variance were used. The read coverage was log transformed. CGs from chromosome 21 were used for detection of T21 markers. Samples from non-pregnant female and pregnant with healthy fetuses women and CV tissue samples were marked as control, whereas only the female samples with T21 positive fetuses were marked as cases. A linear regression model was fitted for every CG, and resulting model fits were moderated using empirical Bayes adjustment. The CGs with FDR q value less than 0.05 and log fold change more than 1.2 were taken as significant. The list of the selected T21 CG-DLRs is shown in Table 6 ( FIG.  4   ). 
     Identification of CG-DLRs for Determination of Fetal Sex. 
     Similarly, CGs from chromosome X (and Y) were analyzed for identification of CG-DLRs for fetal gender determination. A no intercept linear regression model was fitted for each CG and a contrast fit was used to determine differences between male and female samples. Resulting model fits were moderated using empirical Bayes adjustment. The CGs with FDR q value less than 0.05 and log fold change more than 1 were taken as significant. The list of the selected gender CG-DLRs is shown in Table 6 ( FIG.  5   ). 
     Example 3. Evaluation of CG-DLRs by qPCR 
     In this example, individual CGs or CG-DLRs identified according to the methodology described in Examples 1 and 2 were used for their validation by qPCR. A flowchart diagram of the methodology is shown in  FIGS.  8   a  and  c   . Several experiments were carried out to analyze and validate the identified DLRs or individual CGs. These experiments include an evaluation of the variability and reproducibility of the labeling intensity among different individuals and among technical replicates. 
     Detection of Fetal Trisomy T21 by qPCR. 
     The difference in labeling intensity at specific CG-DLRs, shown in Table 6, was tested in blood samples of pregnant female carrying healthy or T21-diagnosed fetuses ( FIG.  6   ). Briefly, DNA of maternal blood sample was treated as described in Example 1. Then, 0.5 ng of the final amplified DNA were used for measurement of the labeling intensity of u-CG-DLRs and hm-CG-DLRs by qPCR with a Rotor-Gene 0 real-time PCR system (Qiagen) using Maxima SybrGreen/ROX qPCR Master Mix (TS). 0.3 mM of each primer pair used in each reaction, wherein one of the primers binds complementarily to a genomic region in close proximity to the CG site (its 5′ end anneals more than 5 nucleotides to the CG being analyzed), and another primer binds in a vicinity of the CG to allow PCR amplification of the region (or selected DLR) to occur. The amplification conditions were set as: 95° C. for 10 min, 40 cycles 95° C. for 15 s, 60° C. for 60 s. 
     In this embodiment, the plurality of CG-DLRs on chromosome 21 comprises one region or a combination of at least two regions, selected from Table 6. The invention also pertains to a composition comprising nucleic acid probes that selectively detect the regions shown in Table 6, preferably, the pair/set of oligonucleotide primers are selected from Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 [First position of the genomic coordinates 
               
               
                 of the selected u-CG-DLRs 
               
               
                 and hm-CG-DLR on chromosome 21 and nucleotide 
               
               
                 sequences of the primers for 
               
               
                 determination of fetal trisomy T21 by qPCR.] 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 PCR 
                   
               
               
                 u-CG-DLR 
                 product, 
                   
               
               
                 coordinate 
                 length 
                 Primer sequence 
               
               
                   
               
               
                 Chr21: 
                 29732020-1, 
                 Seq ID 1: 
               
               
                 29732020 
                 109 bp 
                 5′CAACTCCCTACAG 
               
               
                   
                   
                 CCCCTTG 
               
               
                   
                   
                 Seq ID 2: 
               
               
                   
                   
                 5′AAATTGCATGATT 
               
               
                   
                   
                 CCCCTGACA 
               
               
                   
               
               
                 Chr21: 
                 29732020-2, 
                 Seq ID 3: 
               
               
                 29732020 
                 67 bp 
                 5′ATGACTGGCTTATTTC 
               
               
                   
                   
                 ACTTAGCATC 
               
               
                   
                   
                 Seq ID 4: 
               
               
                   
                   
                 5′AGTCCTGCTATATGCA 
               
               
                   
                   
                 ACACCTT 
               
               
                   
               
               
                 Chr21: 
                 33462648, 
                 Seq ID 5: 
               
               
                 33462648 
                 97 bp 
                 5′GGTATTTACAAAAGT 
               
               
                   
                   
                 CTGCACCTTAGTC 
               
               
                   
                   
                 Seq ID 6: 
               
               
                   
                   
                 5′CTGCCAACTTCACCC 
               
               
                   
                   
                 AGAGT 
               
               
                   
               
               
                 Chr21: 
                 34672959, 
                 Seq ID 7: 
               
               
                 34672959 
                 73 bp 
                 5′TAGAAATCTTTAGGA 
               
               
                   
                   
                 GGTGGTGAATG 
               
               
                   
                   
                 Seq ID 8: 
               
               
                   
                   
                 5′CATGGTGGAAGAGAT 
               
               
                   
                   
                 GGGC 
               
               
                   
               
               
                   
                 PCR 
                   
               
               
                 hm-CG-DLR 
                 product, 
                   
               
               
                 coordinate 
                 length 
                 Primer sequence 
               
               
                   
               
               
                 Chr21: 
                 30341466, 
                 Seq ID 9: 
               
               
                 30341466 
                 101 bp 
                 5′GCAGAGGTTGCAG 
               
               
                   
                   
                 TGAGCTG 
               
               
                   
                   
                 Seq ID 10: 
               
               
                   
                   
                 5′GTCTGGATGCAAAA 
               
               
                   
                   
                 ATCCCTTT 
               
               
                   
               
               
                 Chr21: 
                 46964859, 
                 Seq ID 11: 
               
               
                 46964859 
                 88 bp 
                 5′GCTGTCCCTGTGGT 
               
               
                   
                   
                 TAAGGTC 
               
               
                   
                   
                 Seq ID 12: 
               
               
                   
                   
                 5′GCCACCACAACAGC 
               
               
                   
                   
                 ACCA 
               
               
                   
               
               
                 Chr21: 
                 44084933, 
                 Seq ID 13: 
               
               
                 44084933 
                 89 bp 
                 5′CCCCATCACCAACT 
               
               
                   
                   
                 TCACTC 
               
               
                   
                   
                 Seq ID 14: 
               
               
                   
                   
                 5′GAAACTGAGTCTC 
               
               
                   
                   
                 TCGCAAGG 
               
               
                   
               
            
           
         
       
     
     Detection of Fetal Gender by qPCR. 
     In another embodiment of the invention, the experimentally acquired value for the presence or availability of labeled CGs is estimated through qPCR, in a total untreated, i.e. non-ligated to adaptors and non-preamplified, maternal blood sample as shown in  FIG.  8   c   , for fetal gender determination. Notably, analysis of the selected CG-DLRs in chromosome X is sufficient for detection of fetal gender. This is only one exemplification of the strategy; the similar strategy may be used for determination of fetal trisomy. 
     Firstly, the difference in the abundance of DLR regions starting at specific CGs shown in Table 6 was tested in the 1st trimester CV tissue DNA of both genders and non-pregnant female blood sample DNA. Then, we mixed CV tissue DNA and non-pregnant female peripheral blood plasma DNA to the ratios 20/80 and 0/100 of the CV and plasma DNA, respectively. 10 ng of each sample mixture were labeled and derivatized with the ODN as described above. Next, 1.5 ng of each sample was analyzed in replicates by qPCR. The coordinates of the u-CG-DLRs on chromosomes X and Y and primers for qPCR are shown in Table 3. 
     
       
         
           
               
             
               
                 TABLE 2=3 
               
               
                   
               
               
                 [First position of the genomic coordinates 
               
               
                 of the selected u-CG-DLRs on chromosomes 
               
               
                 X and Y and nucleotide sequences of the 
               
               
                 primers for determination of 
               
               
                 fetal gender by qPCR.] 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 PCR 
                   
               
               
                 u-CG-DLR 
                 product 
                   
               
               
                 coordinate 
                 length 
                 Primer sequence 
               
               
                   
               
               
                 ChrX: 
                 160 bp 
                 Seq ID 15: 5′-CCTCTCTATGGGCAGT 
               
               
                 138802516 
                   
                 CGGTGATTGACCTGCTTCCTGTGTTGAGC 
               
               
                   
                   
                 Seq ID 16: 5′-TGTTACATCTGGTAGT 
               
               
                   
                   
                 CAGTCTCCAAACCACACAAAAAAGTGGAG 
               
               
                   
               
               
                 ChrY: 
                 123 bp 
                 Seq ID 17: 5′-GTAGAAAAAAGTAGA 
               
               
                 14774154 
                   
                 AACAGCAAGGGGAAG 
               
               
                   
                   
                 Seq ID 18:5-TGTTACATCTGGTAGTC 
               
               
                   
                   
                 AGTCTCCAAACCACACAAAAAAGCCCCT 
               
               
                   
               
            
           
         
       
     
     In more detail, DNA of each sample were labeled with eM.Sssl MTase in the presence of 200 μM Ado-6-azide cofactor for 1 hour at 30° C. as described in Example 1 followed by column purification (Oligo Clean&amp;Concentrator-5, Zymo Research). Then, DNA eluted in 8 ul of Elution Buffer was supplemented with 20 uM alkyne DNA oligonucleotide (ODN, 5′-T(alkyneU)TTTTGTGTGGTTTGGAGACTGACTACCAGATGTAACA), the mixture of 8 mM CuBr and 24 mM of THPTA (Sigma) in 50% of DMSO, incubated for 20 min at 45° C. and subsequently diluted to &lt;1.5% DMSO before purification through the GeneJET NGS Cleanup kit (TS). 1.5 ng of the purified DNA were used for measurement of the labeling intensity of uCGs by qPCR with a Rotor-GeneQ real-time PCR system (Qiagen) using Maxima SybrGreen/ROX qPCR Master Mix (TS). 0.3 mM of each primer pair was used in each reaction, wherein one of the primers binds complementarily to the ODN and to 5 nucleotides of the template genomic DNA adjacent to the derivatized CG site, and another primer binds in a vicinity of the CG to allow PCR amplification of the region (or selected DLR) to occur. The amplification program was set as: 95° C. for 10 min, 40 cycles 95° C. for 15 s, 65° C. for 30 s, 72° C. for 30 s ( FIG.  7     a,b,c ). 
     Example 4. qPCR-Based Noninvasive Diagnostics of Trisomy 21 
     This example describes the independent validation of non-invasive testing for fetal trisomy 21. For this purpose, we have performed qPCR-based analysis of a small group of samples which have not been used in the previous Examples for identification of validation of DLRs. The group consists of 3 maternal peripheral blood samples from women bearing a normal fetus and 2 maternal peripheral blood samples from women bearing a trisomy 21-affected fetus. 
     These maternal peripheral blood samples were obtained at a gestational age of between 12-13 weeks at Tartu University Hospital (Tartu, Estonia) through collaboration with Tartu University (Estonia). Consent forms approved by the Research Ethics Committee of the University of Tartu (ethical permission No. 246/T-21 and 213/T-21) were collected for each of the mother participated. 
     The fetal specific approach used herein is illustrated schematically in  FIG.  8   a   , wherein the ability to discriminate normal from trisomy 21 cases is achieved by comparing the values obtained from normal and trisomy 21 cases using T21-specific differentially modified CG dinucleotides, or CG-DLRs, located on chromosome 21. A fetus with trisomy 21 has a differentially modified genome in relation to normal genome and an extra copy of chromosome 21, and thus the increased abundance of a fetal specific region compared to a normal fetus. Therefore, the amount of T21-specific fetal region will increase more in fetuses with trisomy 21 compared to normal cases. 
     An in-depth investigation of our previously identified DLRs, described in Examples 1 and 2, has led to selection of DLRs located on chromosome 21. A group of selected DLRs has been used for identification of fetal trisomy 21 by qPCR (Example 3). These DLRs demonstrate a hypomethylated or hyper-hydroxymethylated, and thus more labeled, status in peripheral blood DNA of pregnant women carrying a T21-diagnosed fetus and a hypermethylated or hypo-hydroxymethylated, and thus less labeled, status in CV tissue DNA and peripheral blood DNA of pregnant women carrying a normal fetus and in peripheral blood DNA of non-pregnant women in order to achieve the enrichment of fetal T21-specific CG-labeled regions. These selected CG-DLRs shown in Table 2 were used for analysis of the samples by qPCR. 
     The procedure of sample processing and qPCR cycle conditions used were as described in Examples 1 and 3. Briefly, 5-10 ng of maternal cfDNA was covalently derivatized with the ODN and the adaptors were ligated to the ends of DNA fragments. The labeled CG regions were enriched through the ODN-mediated polymerization of the adjacent genomic regions and such regions were subsequently amplified using the primers complementary to the ODN and one strand of the adaptors. Then, the amounts of u-CG-DLRs and hm-CG-DLRs was calculated by qPCR as shown in Example 3 using a combination of CG-DLRs and qPCR primers listed in Table 2. 
     Comparing the obtained test values of the samples with known karyotype (the T21-diagnosed samples show lower test values than normal cases), all T21-diagnosed samples were confirmed as having trisomy 21, indicating 100% specificity and 100% sensitivity of the approach ( FIG.  10   ). 
     APPENDICES 
       
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 [The coordinate is shown for the first base pair of 100 bp u-DLRs in chromosome 21] 
               
               
                   
               
             
            
               
                 Pregnancy-specific u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10774500 
                 26212900 
                 35812700 
                 38891600 
                 43228800 
                 45323700 
                 46743900 
                 47331000 
               
               
                 11025700 
                 26835100 
                 35819500 
                 38946900 
                 43470300 
                 45330400 
                 46751000 
                 47331900 
               
               
                 15169700 
                 28041300 
                 35879100 
                 38969700 
                 43519400 
                 45355100 
                 46808700 
                 47362600 
               
               
                 15770300 
                 28074300 
                 36073900 
                 39202100 
                 43708100 
                 45392900 
                 46812700 
                 47390300 
               
               
                 16130900 
                 28759100 
                 36089600 
                 39507100 
                 43714600 
                 45400400 
                 46837800 
                 47419000 
               
               
                 16577200 
                 28942700 
                 36220800 
                 39544400 
                 43728400 
                 45597600 
                 46847100 
                 47451100 
               
               
                 17308600 
                 29288000 
                 36437300 
                 39690100 
                 43782100 
                 45734900 
                 46934400 
                 47479200 
               
               
                 17333200 
                 31008200 
                 36478700 
                 39891300 
                 43864600 
                 45748300 
                 46946300 
                 47498400 
               
               
                 18086100 
                 32374100 
                 36701300 
                 41001100 
                 43864800 
                 45753500 
                 46973100 
                 47502800 
               
               
                 18676300 
                 32639100 
                 36917100 
                 41292800 
                 43876000 
                 45790600 
                 46995500 
                 47536100 
               
               
                 18940600 
                 32915800 
                 37085900 
                 42099000 
                 44061700 
                 45842200 
                 46997700 
                 47542700 
               
               
                 20437200 
                 33522600 
                 37192500 
                 42127100 
                 44113000 
                 46036100 
                 46999800 
                 47549500 
               
               
                 20608700 
                 33533900 
                 37218700 
                 42212900 
                 44191100 
                 46182600 
                 47057200 
                 47559700 
               
               
                 21354200 
                 33591700 
                 37352800 
                 42424900 
                 44196200 
                 46312000 
                 47181700 
                 48047900 
               
               
                 21670800 
                 33954100 
                 37493000 
                 42595400 
                 44208900 
                 46359300 
                 47211600 
                 48079600 
               
               
                 22564300 
                 34369300 
                 37527800 
                 42694800 
                 44346000 
                 46396600 
                 47212000 
                 9901200 
               
               
                 24387600 
                 34406400 
                 37970500 
                 42732500 
                 44474700 
                 46415700 
                 47213500 
               
               
                 24474800 
                 34483300 
                 38066600 
                 42746400 
                 44511200 
                 46418600 
                 47245100 
               
               
                 25233800 
                 34851100 
                 38092400 
                 42928900 
                 44754300 
                 46545400 
                 47273200 
               
               
                 25693500 
                 35365900 
                 38104700 
                 42936000 
                 45065800 
                 46720900 
                 47287600 
               
               
                 26152100 
                 35531600 
                 38385400 
                 43112600 
                 45156300 
                 46738900 
                 47315300 
               
            
           
           
               
            
               
                 Tissue-specific u-DLRs. Only 1000 selected DLRs are shown 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10027900 
                 15984000 
                 17333200 
                 18351000 
                 19378400 
                 20630400 
                 21741100 
                 22819200 
               
               
                 10395200 
                 15993500 
                 17333300 
                 18356700 
                 19379400 
                 20633700 
                 21743700 
                 22820400 
               
               
                 10527800 
                 16003800 
                 17344200 
                 18361400 
                 19382400 
                 20655700 
                 21745700 
                 22830600 
               
               
                 10551600 
                 16009000 
                 17364800 
                 18387200 
                 19390200 
                 20668400 
                 21746900 
                 22842800 
               
               
                 10603000 
                 16010900 
                 17377300 
                 18389400 
                 19391100 
                 20685000 
                 21755000 
                 22848600 
               
               
                 10713200 
                 16015300 
                 17382300 
                 18399000 
                 19392100 
                 20698000 
                 21765500 
                 22866100 
               
               
                 10757400 
                 16016600 
                 17384200 
                 18418800 
                 19392900 
                 20701500 
                 21771800 
                 22880300 
               
               
                 10762300 
                 16025200 
                 17389700 
                 18426600 
                 19397200 
                 20706300 
                 21775600 
                 22896500 
               
               
                 10762500 
                 16033800 
                 17392100 
                 18433700 
                 19400200 
                 20715200 
                 21775700 
                 22925400 
               
               
                 10807400 
                 16039700 
                 17396700 
                 18444200 
                 19406900 
                 20719900 
                 21802300 
                 22926000 
               
               
                 10812800 
                 16046800 
                 17400700 
                 18449500 
                 19415500 
                 20748600 
                 21809600 
                 22926600 
               
               
                 10821600 
                 16051800 
                 17405400 
                 18461000 
                 19427200 
                 20749700 
                 21814100 
                 22936200 
               
               
                 10824800 
                 16056200 
                 17405500 
                 18483000 
                 19427900 
                 20759800 
                 21826100 
                 22947400 
               
               
                 10826000 
                 16058300 
                 17422500 
                 18492200 
                 19429700 
                 20763100 
                 21831900 
                 22970300 
               
               
                 10836600 
                 16065200 
                 17423000 
                 18497900 
                 19432000 
                 20780900 
                 21832100 
                 22975500 
               
               
                 10851100 
                 16065400 
                 17423200 
                 18519100 
                 19443000 
                 20790400 
                 21838500 
                 22981800 
               
               
                 10851700 
                 16066400 
                 17434500 
                 18527800 
                 19443800 
                 20806100 
                 21840500 
                 22984400 
               
               
                 10862500 
                 16076200 
                 17440300 
                 18535800 
                 19486900 
                 20808400 
                 21850900 
                 23000600 
               
               
                 10868300 
                 16087900 
                 17443100 
                 18550100 
                 19495300 
                 20814400 
                 21851100 
                 23009700 
               
               
                 10889500 
                 16099900 
                 17456000 
                 18570400 
                 19495400 
                 20825700 
                 21851800 
                 23012900 
               
               
                 10898800 
                 16104500 
                 17461800 
                 18587100 
                 19496300 
                 20834300 
                 21852800 
                 23032500 
               
               
                 10990600 
                 16105700 
                 17464000 
                 18603400 
                 19501800 
                 20867700 
                 21852900 
                 23058900 
               
               
                 11021600 
                 16120400 
                 17464100 
                 18611400 
                 19506400 
                 20869900 
                 21856600 
                 23061100 
               
               
                 11025700 
                 16127400 
                 17466600 
                 18618800 
                 19508200 
                 20876400 
                 21883000 
                 23061600 
               
               
                 11034800 
                 16130900 
                 17466800 
                 18619900 
                 19514500 
                 20876600 
                 21888700 
                 23094800 
               
               
                 11048000 
                 16141200 
                 17467500 
                 18622300 
                 19523800 
                 20889300 
                 21891500 
                 23095300 
               
               
                 11096200 
                 16151900 
                 17481900 
                 18634200 
                 19526200 
                 20893200 
                 21892000 
                 23101600 
               
               
                 11100600 
                 16159500 
                 17505400 
                 18637600 
                 19526700 
                 20898100 
                 21893600 
                 23126800 
               
               
                 11106600 
                 16163900 
                 17506600 
                 18643800 
                 19530700 
                 20900800 
                 21900500 
                 23129400 
               
               
                 11127600 
                 16176600 
                 17517700 
                 18668800 
                 19531400 
                 20903700 
                 21928500 
                 23185500 
               
               
                 11153500 
                 16182300 
                 17519700 
                 18676300 
                 19552600 
                 20912300 
                 21935000 
                 23191700 
               
               
                 11161300 
                 16218500 
                 17528400 
                 18677300 
                 19562100 
                 20920500 
                 21938500 
                 23196100 
               
               
                 11180200 
                 16229300 
                 17532700 
                 18678900 
                 19569200 
                 20930800 
                 21940100 
                 23235500 
               
               
                 14344600 
                 16259600 
                 17561300 
                 18685400 
                 19569800 
                 20941500 
                 21950400 
                 23236400 
               
               
                 14361600 
                 16260700 
                 17561800 
                 18699800 
                 19591300 
                 20944400 
                 21965200 
                 23240500 
               
               
                 14372500 
                 16288000 
                 17573800 
                 18707300 
                 19596200 
                 20956500 
                 21978100 
                 23275900 
               
               
                 14383200 
                 16291200 
                 17582800 
                 18707600 
                 19603500 
                 20967800 
                 21988500 
                 23276000 
               
               
                 14390500 
                 16307500 
                 17584600 
                 18715900 
                 19613900 
                 21013800 
                 22001400 
                 23290600 
               
               
                 14395600 
                 16396400 
                 17586100 
                 18716200 
                 19615600 
                 21024900 
                 22018800 
                 23296900 
               
               
                 14411800 
                 16443400 
                 17595800 
                 18720000 
                 19616500 
                 21050300 
                 22031600 
                 23303600 
               
               
                 14431400 
                 16452000 
                 17619000 
                 18740600 
                 19619500 
                 21074500 
                 22043300 
                 23326200 
               
               
                 14699500 
                 16458600 
                 17620100 
                 18741900 
                 19635700 
                 21081100 
                 22060800 
                 23328300 
               
               
                 14805600 
                 16461400 
                 17621100 
                 18748500 
                 19643700 
                 21091600 
                 22062000 
                 23328700 
               
               
                 14828200 
                 16518900 
                 17627600 
                 18778900 
                 19649100 
                 21101600 
                 22080100 
                 23338700 
               
               
                 14897900 
                 16520800 
                 17633400 
                 18783100 
                 19649900 
                 21104100 
                 22086700 
                 23341100 
               
               
                 14900100 
                 16528100 
                 17635100 
                 18788000 
                 19657900 
                 21104300 
                 22105900 
                 23345000 
               
               
                 14944200 
                 16553000 
                 17637900 
                 18797600 
                 19688500 
                 21108700 
                 22106200 
                 23354800 
               
               
                 14950900 
                 16556500 
                 17643000 
                 18798400 
                 19708400 
                 21110400 
                 22115900 
                 23356100 
               
               
                 15036300 
                 16558500 
                 17663200 
                 18800100 
                 19719700 
                 21136800 
                 22133500 
                 23360300 
               
               
                 15054200 
                 16565200 
                 17666500 
                 18819100 
                 19727600 
                 21140800 
                 22134300 
                 23365200 
               
               
                 15078000 
                 16569700 
                 17670500 
                 18821500 
                 19731400 
                 21143400 
                 22134400 
                 23372200 
               
               
                 15083000 
                 16582300 
                 17683800 
                 18831800 
                 19738100 
                 21149400 
                 22138200 
                 23382900 
               
               
                 15087900 
                 16604700 
                 17698700 
                 18834000 
                 19743000 
                 21151800 
                 22144800 
                 23389900 
               
               
                 15141900 
                 16606800 
                 17703300 
                 18836800 
                 19756000 
                 21158900 
                 22145200 
                 23401700 
               
               
                 15194400 
                 16614900 
                 17707500 
                 18839500 
                 19757000 
                 21160100 
                 22159500 
                 23403000 
               
               
                 15255400 
                 16628500 
                 17709400 
                 18848600 
                 19759100 
                 21167300 
                 22161000 
                 23404600 
               
               
                 15323900 
                 16629000 
                 17710800 
                 18857500 
                 19763800 
                 21172800 
                 22171200 
                 23405000 
               
               
                 15356300 
                 16633000 
                 17752400 
                 18880600 
                 19786900 
                 21174500 
                 22173800 
                 23405200 
               
               
                 15372700 
                 16643000 
                 17758800 
                 18900200 
                 19794500 
                 21183100 
                 22174700 
                 23407500 
               
               
                 15398100 
                 16644600 
                 17759500 
                 18907500 
                 19795600 
                 21186600 
                 22218100 
                 23426000 
               
               
                 15412500 
                 16654100 
                 17784400 
                 18909300 
                 19824300 
                 21189400 
                 22224000 
                 23456500 
               
               
                 15434900 
                 16685000 
                 17788300 
                 18910100 
                 19824700 
                 21189800 
                 22234100 
                 23467900 
               
               
                 15435600 
                 16686400 
                 17813400 
                 18914600 
                 19825200 
                 21192600 
                 22243700 
                 23490200 
               
               
                 15445400 
                 16694900 
                 17827300 
                 18918900 
                 19831700 
                 21223200 
                 22255400 
                 23492700 
               
               
                 15451600 
                 16707300 
                 17828200 
                 18921800 
                 19837300 
                 21224300 
                 22264200 
                 23502200 
               
               
                 15528500 
                 16732500 
                 17832300 
                 18942200 
                 19849300 
                 21226200 
                 22265000 
                 23507500 
               
               
                 15546200 
                 16744000 
                 17838700 
                 18943400 
                 19858300 
                 21233900 
                 22270700 
                 23510800 
               
               
                 15576100 
                 16756800 
                 17856200 
                 18951300 
                 19859200 
                 21238500 
                 22278500 
                 23534900 
               
               
                 15589600 
                 16777100 
                 17864500 
                 18955000 
                 19865900 
                 21239300 
                 22280600 
                 23576400 
               
               
                 15607200 
                 16786100 
                 17864900 
                 18956800 
                 19867200 
                 21273800 
                 22281500 
                 23582100 
               
               
                 15608600 
                 16798600 
                 17875800 
                 18998400 
                 19890600 
                 21282600 
                 22281800 
                 23611500 
               
               
                 15609000 
                 16859000 
                 17877800 
                 18999200 
                 19891100 
                 21283300 
                 22294100 
                 23657500 
               
               
                 15615500 
                 16860400 
                 17885300 
                 19000900 
                 19903500 
                 21301000 
                 22312200 
                 23659400 
               
               
                 15620000 
                 16863700 
                 17896400 
                 19010800 
                 19911700 
                 21304400 
                 22313200 
                 23667100 
               
               
                 15629000 
                 16872300 
                 17898500 
                 19023900 
                 19912700 
                 21310100 
                 22319300 
                 23672700 
               
               
                 15629700 
                 16875900 
                 17914500 
                 19026000 
                 19940300 
                 21316200 
                 22323700 
                 23696400 
               
               
                 15630300 
                 16885500 
                 17916200 
                 19030300 
                 19952500 
                 21326500 
                 22325600 
                 23718600 
               
               
                 15633600 
                 16892300 
                 17935900 
                 19041100 
                 19979900 
                 21333300 
                 22331400 
                 23779300 
               
               
                 15639900 
                 16894100 
                 17939600 
                 19044700 
                 19985200 
                 21354100 
                 22333100 
                 23793400 
               
               
                 15641300 
                 16904300 
                 17961100 
                 19047700 
                 20009000 
                 21354200 
                 22337300 
                 23803600 
               
               
                 15646800 
                 16922200 
                 17976600 
                 19049500 
                 20015100 
                 21365300 
                 22337800 
                 23808300 
               
               
                 15650300 
                 16932600 
                 17982200 
                 19053900 
                 20024300 
                 21366400 
                 22347600 
                 23826100 
               
               
                 15665500 
                 16934400 
                 17985200 
                 19054300 
                 20120500 
                 21381000 
                 22347900 
                 23831100 
               
               
                 15670200 
                 16936900 
                 17997200 
                 19063100 
                 20120600 
                 21388800 
                 22351600 
                 23833100 
               
               
                 15673600 
                 16942700 
                 18009700 
                 19082400 
                 20128100 
                 21400400 
                 22356700 
                 23833700 
               
               
                 15676400 
                 16943100 
                 18023100 
                 19095100 
                 20131000 
                 21404000 
                 22371200 
                 23833900 
               
               
                 15686900 
                 16963300 
                 18032400 
                 19098100 
                 20131500 
                 21415000 
                 22378400 
                 23835900 
               
               
                 15687000 
                 16964400 
                 18038700 
                 19100300 
                 20149600 
                 21416100 
                 22381400 
                 23840200 
               
               
                 15703200 
                 16967500 
                 18040100 
                 19108000 
                 20180700 
                 21416900 
                 22383100 
                 23853500 
               
               
                 15709200 
                 16969300 
                 18049300 
                 19110700 
                 20200400 
                 21433900 
                 22385800 
                 23859400 
               
               
                 15709900 
                 16979600 
                 18054800 
                 19114200 
                 20208800 
                 21436900 
                 22392900 
                 23871200 
               
               
                 15713500 
                 16996400 
                 18077000 
                 19117100 
                 20227000 
                 21454900 
                 22402900 
                 23876400 
               
               
                 15715700 
                 16997200 
                 18086100 
                 19117200 
                 20239300 
                 21460100 
                 22423000 
                 23877000 
               
               
                 15717600 
                 17008700 
                 18097800 
                 19117800 
                 20239400 
                 21467700 
                 22456400 
                 23880000 
               
               
                 15724200 
                 17012700 
                 18102600 
                 19117900 
                 20260500 
                 21475300 
                 22459000 
                 23890200 
               
               
                 15741400 
                 17017800 
                 18109800 
                 19119800 
                 20268300 
                 21476300 
                 22462200 
                 23890500 
               
               
                 15757600 
                 17025900 
                 18118400 
                 19132800 
                 20286400 
                 21482800 
                 22477300 
                 23891200 
               
               
                 15760000 
                 17030000 
                 18127000 
                 19147700 
                 20289300 
                 21490300 
                 22478300 
                 23901600 
               
               
                 15770300 
                 17062200 
                 18145400 
                 19203900 
                 20295300 
                 21493900 
                 22482400 
                 23917700 
               
               
                 15782400 
                 17063900 
                 18154600 
                 19205400 
                 20320600 
                 21496200 
                 22495500 
                 23918900 
               
               
                 15791300 
                 17064700 
                 18157800 
                 19205800 
                 20322700 
                 21496700 
                 22498300 
                 23923900 
               
               
                 15806500 
                 17074500 
                 18163100 
                 19205900 
                 20326300 
                 21499100 
                 22519200 
                 23931300 
               
               
                 15807300 
                 17079000 
                 18168000 
                 19206700 
                 20337600 
                 21501000 
                 22534100 
                 23934300 
               
               
                 15810000 
                 17086600 
                 18169800 
                 19218500 
                 20349500 
                 21501100 
                 22547500 
                 23941700 
               
               
                 15811800 
                 17090900 
                 18172800 
                 19219800 
                 20353400 
                 21509600 
                 22562200 
                 23950900 
               
               
                 15820300 
                 17094100 
                 18179000 
                 19221900 
                 20356400 
                 21516800 
                 22582000 
                 23951200 
               
               
                 15833000 
                 17101300 
                 18187900 
                 19222000 
                 20362800 
                 21522400 
                 22606900 
                 23952600 
               
               
                 15834600 
                 17103200 
                 18196800 
                 19223900 
                 20364900 
                 21533000 
                 22607100 
                 23959700 
               
               
                 15838300 
                 17111700 
                 18200900 
                 19248800 
                 20394300 
                 21548500 
                 22615800 
                 23972500 
               
               
                 15839100 
                 17116600 
                 18217200 
                 19251000 
                 20395900 
                 21567700 
                 22631300 
                 23981400 
               
               
                 15845600 
                 17133800 
                 18221600 
                 19252500 
                 20429600 
                 21594700 
                 22646300 
                 23983100 
               
               
                 15853600 
                 17154600 
                 18223800 
                 19256700 
                 20436900 
                 21616500 
                 22687500 
                 23992300 
               
               
                 15866100 
                 17160400 
                 18233400 
                 19274300 
                 20453600 
                 21636700 
                 22690100 
                 23999500 
               
               
                 15876600 
                 17207100 
                 18251500 
                 19288100 
                 20462000 
                 21636900 
                 22697000 
                 24018400 
               
               
                 15882100 
                 17218700 
                 18252100 
                 19288200 
                 20476500 
                 21637100 
                 22701400 
                 24018800 
               
               
                 15899800 
                 17276800 
                 18259400 
                 19296000 
                 20500600 
                 21643600 
                 22744100 
                 24025300 
               
               
                 15909400 
                 17278400 
                 18267400 
                 19302000 
                 20506900 
                 21670800 
                 22745700 
                 24057800 
               
               
                 15928100 
                 17279200 
                 18274100 
                 19305800 
                 20519200 
                 21672100 
                 22754900 
                 24061000 
               
               
                 15936100 
                 17285600 
                 18301200 
                 19317400 
                 20536400 
                 21678300 
                 22762300 
                 24074500 
               
               
                 15941400 
                 17292700 
                 18310100 
                 19328300 
                 20548000 
                 21678700 
                 22769500 
                 24084900 
               
               
                 15947900 
                 17296700 
                 18315000 
                 19334800 
                 20566900 
                 21679300 
                 22773000 
                 24089300 
               
               
                 15955700 
                 17300600 
                 18317500 
                 19335700 
                 20581300 
                 21691700 
                 22790800 
                 24105100 
               
               
                 15970000 
                 17303600 
                 18322500 
                 19346600 
                 20591500 
                 21714200 
                 22809100 
                 24113400 
               
               
                 15972100 
                 17315700 
                 18325500 
                 19354400 
                 20608700 
                 21719300 
                 22809300 
                 24114000 
               
               
                 15982800 
                 17320300 
                 18334900 
                 19375100 
                 20614800 
                 21739500 
                 22818500 
                 24139100 
               
            
           
           
               
            
               
                 The selected T21-specific u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 15078000 
                 20843100 
                 24937300 
                 31821900 
                 34672900 
                 42770000 
                 47588600 
                 38660800 
               
               
                 15413700 
                 21451400 
                 25752700 
                 32258800 
                 34690700 
                 43291800 
                 9875200 
                 41842500 
               
               
                 15486300 
                 21739100 
                 25887700 
                 32294400 
                 34872200 
                 43644200 
                 18679500 
                 45355100 
               
               
                 15490100 
                 21771600 
                 26081000 
                 32526200 
                 35234300 
                 43933400 
                 22295500 
                 45734900 
               
               
                 15680600 
                 22449400 
                 28463100 
                 32748600 
                 36191800 
                 44303300 
                 22450800 
                 45770300 
               
               
                 16547900 
                 22459500 
                 29713600 
                 32900300 
                 36193500 
                 44303600 
                 26152100 
                 45946100 
               
               
                 17461600 
                 22530700 
                 29732000 
                 33572800 
                 37070300 
                 45151100 
                 31408200 
                 46316400 
               
               
                 18123400 
                 22715800 
                 29879100 
                 33831600 
                 38032500 
                 45597700 
                 32639100 
                 48079600 
               
               
                 18499700 
                 22908900 
                 31306700 
                 33875700 
                 39652400 
                 45708400 
                 33533900 
               
               
                 19286900 
                 22921700 
                 31357700 
                 33919200 
                 40405900 
                 46009400 
                 33591700 
               
               
                 20037100 
                 23004300 
                 31489300 
                 34092400 
                 41285400 
                 46780500 
                 33840400 
               
               
                 20042500 
                 23380000 
                 31568000 
                 34460800 
                 42378900 
                 47329500 
                 36220800 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 [The coordinate is shown for the first base pair of 100 bp hm-DLRs in chromosome 21] 
               
               
                   
               
             
            
               
                 Pregnancy-specific hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 15078000 
                 30594600 
                 35231100 
                 38321300 
                 42695100 
                 44301100 
                 45492400 
                 46747600 
               
               
                 15442500 
                 30642600 
                 35246100 
                 38335200 
                 42738300 
                 44326600 
                 45494400 
                 46748200 
               
               
                 15496700 
                 30658000 
                 35272400 
                 38441400 
                 42746900 
                 44329100 
                 45498400 
                 46748800 
               
               
                 15970900 
                 30669700 
                 35293400 
                 38443100 
                 42760300 
                 44334600 
                 45542100 
                 46769800 
               
               
                 16119600 
                 30675000 
                 35344900 
                 38454300 
                 42824800 
                 44347000 
                 45560600 
                 46776900 
               
               
                 16193000 
                 30708600 
                 35349600 
                 38541100 
                 42851600 
                 44350500 
                 45568800 
                 46777600 
               
               
                 16213800 
                 30719600 
                 35444700 
                 38566700 
                 42860000 
                 44354200 
                 45571400 
                 46780700 
               
               
                 16214600 
                 30755900 
                 35500600 
                 38567300 
                 42860500 
                 44361200 
                 45572100 
                 46784600 
               
               
                 16240400 
                 31030700 
                 35516100 
                 38579100 
                 42874000 
                 44366300 
                 45614500 
                 46790700 
               
               
                 16311800 
                 31223600 
                 35560500 
                 38634900 
                 43030200 
                 44381700 
                 45621700 
                 46799200 
               
               
                 16326500 
                 32471400 
                 35587700 
                 38636200 
                 43050800 
                 44383700 
                 45630000 
                 46869900 
               
               
                 16389000 
                 32510000 
                 35616700 
                 38662600 
                 43092100 
                 44387300 
                 45632500 
                 46870200 
               
               
                 16395200 
                 32575500 
                 35712600 
                 38676800 
                 43115200 
                 44387900 
                 45637600 
                 46886600 
               
               
                 16396200 
                 32581400 
                 35718200 
                 38732000 
                 43135400 
                 44442400 
                 45658000 
                 46902700 
               
               
                 16407900 
                 32678200 
                 35755000 
                 38750600 
                 43154800 
                 44448300 
                 45659300 
                 46911600 
               
               
                 16488800 
                 32711300 
                 35761500 
                 38766900 
                 43171100 
                 44461800 
                 45663300 
                 46914600 
               
               
                 16511900 
                 32725600 
                 35879600 
                 38767300 
                 43172900 
                 44467700 
                 45675100 
                 46924300 
               
               
                 16572400 
                 32831900 
                 35884200 
                 38822100 
                 43175500 
                 44475900 
                 45704700 
                 46925400 
               
               
                 16572800 
                 32840800 
                 35886800 
                 38832800 
                 43175800 
                 44491400 
                 45705900 
                 46931500 
               
               
                 16582800 
                 32898600 
                 35893700 
                 38888500 
                 43179100 
                 44508400 
                 45724700 
                 46932100 
               
               
                 16591800 
                 32915200 
                 35894400 
                 38890600 
                 43228700 
                 44573600 
                 45743000 
                 46932200 
               
               
                 16643600 
                 32915700 
                 35922100 
                 38920900 
                 43228800 
                 44591600 
                 45747000 
                 46932700 
               
               
                 16682300 
                 32934700 
                 35940800 
                 38942100 
                 43239800 
                 44594100 
                 45751700 
                 46934200 
               
               
                 16684400 
                 32986500 
                 35963200 
                 38964000 
                 43241000 
                 44595900 
                 45773900 
                 46945700 
               
               
                 16706000 
                 32999300 
                 36072400 
                 39104900 
                 43242000 
                 44614600 
                 45796000 
                 46950400 
               
               
                 16763200 
                 33005100 
                 36076400 
                 39343900 
                 43245800 
                 44626800 
                 45825100 
                 46959000 
               
               
                 16828600 
                 33012700 
                 36079700 
                 39461200 
                 43256600 
                 44704800 
                 45826500 
                 46959800 
               
               
                 16884200 
                 33019200 
                 36081500 
                 39490900 
                 43293000 
                 44732600 
                 45843300 
                 46971900 
               
               
                 16888000 
                 33026800 
                 36108600 
                 39594800 
                 43314600 
                 44762100 
                 45880200 
                 46973300 
               
               
                 17036800 
                 33057900 
                 36157900 
                 39598900 
                 43319100 
                 44782500 
                 45883300 
                 46977800 
               
               
                 17086600 
                 33085600 
                 36164200 
                 39632900 
                 43319500 
                 44784900 
                 45898800 
                 46980800 
               
               
                 17099100 
                 33671200 
                 36165000 
                 39706900 
                 43344400 
                 44802600 
                 45928000 
                 47051900 
               
               
                 17099800 
                 33723100 
                 36198900 
                 39755900 
                 43351200 
                 44814600 
                 45956000 
                 47124500 
               
               
                 17117000 
                 33725000 
                 36202900 
                 39761300 
                 43376500 
                 44817100 
                 46034900 
                 47188800 
               
               
                 17193500 
                 33763500 
                 36208100 
                 39851400 
                 43384100 
                 44837100 
                 46055600 
                 47239300 
               
               
                 17550000 
                 33792500 
                 36242900 
                 39948700 
                 43394500 
                 44870700 
                 46063700 
                 47251700 
               
               
                 17561300 
                 33805800 
                 36243000 
                 39970500 
                 43412800 
                 44871500 
                 46068700 
                 47288700 
               
               
                 17578000 
                 33823700 
                 36244900 
                 39984000 
                 43443300 
                 44872300 
                 46142700 
                 47290300 
               
               
                 17592700 
                 33857800 
                 36246600 
                 40119400 
                 43446200 
                 44876300 
                 46154200 
                 47333300 
               
               
                 17666600 
                 33881100 
                 36288500 
                 40123900 
                 43456200 
                 44883100 
                 46182800 
                 47403100 
               
               
                 17734100 
                 33901800 
                 36329200 
                 40134200 
                 43499900 
                 44883700 
                 46214100 
                 47418400 
               
               
                 18846300 
                 33948600 
                 36331900 
                 40166900 
                 43506100 
                 44900600 
                 46235600 
                 47422300 
               
               
                 18857600 
                 33957500 
                 36345600 
                 40176200 
                 43567000 
                 44916900 
                 46253400 
                 47423800 
               
               
                 18883100 
                 33966100 
                 36389700 
                 40244600 
                 43571300 
                 44924000 
                 46270000 
                 47457000 
               
               
                 19052700 
                 34062900 
                 36444900 
                 40277900 
                 43577500 
                 44928000 
                 46271300 
                 47515500 
               
               
                 19066500 
                 34069100 
                 36595800 
                 40285300 
                 43603000 
                 44928800 
                 46272800 
                 47520800 
               
               
                 19069400 
                 34075400 
                 36656600 
                 40293700 
                 43621300 
                 44935600 
                 46284800 
                 47530400 
               
               
                 19071700 
                 34185900 
                 36693800 
                 40293800 
                 43679800 
                 45039800 
                 46286600 
                 47538400 
               
               
                 19106400 
                 34338600 
                 36829500 
                 40310600 
                 43681500 
                 45040100 
                 46307200 
                 47541800 
               
               
                 19118800 
                 34402600 
                 36840000 
                 40349000 
                 43786800 
                 45064600 
                 46308100 
                 47542400 
               
               
                 19150100 
                 34405900 
                 36944400 
                 40352500 
                 43790900 
                 45067700 
                 46319500 
                 47545100 
               
               
                 19173600 
                 34409600 
                 37015500 
                 40356600 
                 43801700 
                 45092200 
                 46320800 
                 47552700 
               
               
                 19176300 
                 34447800 
                 37033700 
                 40357800 
                 43813600 
                 45105500 
                 46326500 
                 47556200 
               
               
                 19228600 
                 34477900 
                 37038600 
                 40358000 
                 43817900 
                 45109700 
                 46328000 
                 47574100 
               
               
                 21311300 
                 34517400 
                 37169900 
                 40372800 
                 43844600 
                 45116700 
                 46349100 
                 47577700 
               
               
                 21626600 
                 34556200 
                 37277600 
                 40394400 
                 43846300 
                 45129900 
                 46371800 
                 47608400 
               
               
                 22421400 
                 34618500 
                 37334100 
                 40395100 
                 43846800 
                 45131700 
                 46379600 
                 47617600 
               
               
                 23735900 
                 34623200 
                 37436800 
                 40453300 
                 43869600 
                 45147000 
                 46396500 
                 47624100 
               
               
                 25184700 
                 34625700 
                 37456000 
                 40466200 
                 43872000 
                 45153200 
                 46398100 
                 47630200 
               
               
                 25711300 
                 34638000 
                 37459800 
                 40466900 
                 43893300 
                 45182000 
                 46401200 
                 47631900 
               
               
                 27006500 
                 34643100 
                 37537100 
                 40479000 
                 43896600 
                 45190800 
                 46403700 
                 47632900 
               
               
                 27157900 
                 34717700 
                 37542300 
                 40479900 
                 43898300 
                 45191200 
                 46407900 
                 47676300 
               
               
                 27190500 
                 34722900 
                 37554900 
                 40542000 
                 43915300 
                 45228200 
                 46412700 
                 47686900 
               
               
                 27287800 
                 34728000 
                 37559100 
                 40568900 
                 43943900 
                 45229000 
                 46442000 
                 47700500 
               
               
                 27332200 
                 34753100 
                 37609600 
                 40637700 
                 43977800 
                 45232900 
                 46449800 
                 47715600 
               
               
                 27397400 
                 34754700 
                 37627900 
                 40730800 
                 43988800 
                 45234200 
                 46451400 
                 47764000 
               
               
                 27424500 
                 34756700 
                 37639400 
                 40741800 
                 44003400 
                 45242100 
                 46455200 
                 47766000 
               
               
                 27434400 
                 34774100 
                 37646900 
                 40763200 
                 44004600 
                 45244300 
                 46455400 
                 47780500 
               
               
                 27445300 
                 34790400 
                 37658400 
                 40773600 
                 44006200 
                 45246200 
                 46461600 
                 47786500 
               
               
                 27449100 
                 34790500 
                 37674700 
                 40815500 
                 44033500 
                 45253700 
                 46473600 
                 47793600 
               
               
                 27452300 
                 34811500 
                 37750300 
                 40841700 
                 44037100 
                 45271700 
                 46480500 
                 47805500 
               
               
                 27489500 
                 34814600 
                 37758700 
                 40881500 
                 44053600 
                 45286500 
                 46491900 
                 47861300 
               
               
                 27559600 
                 34848800 
                 37772200 
                 41010600 
                 44064900 
                 45298300 
                 46560000 
                 47939900 
               
               
                 27895000 
                 34911100 
                 37791900 
                 41086000 
                 44075700 
                 45298700 
                 46566300 
                 47946100 
               
               
                 27938500 
                 34923100 
                 37795000 
                 41130500 
                 44115900 
                 45299700 
                 46568900 
                 47976300 
               
               
                 28256200 
                 35023200 
                 37819200 
                 41132500 
                 44117300 
                 45325300 
                 46640200 
                 47980600 
               
               
                 28307900 
                 35047800 
                 37978700 
                 41919500 
                 44144600 
                 45338100 
                 46643200 
                 47983300 
               
               
                 28515800 
                 35058800 
                 38028500 
                 42036400 
                 44152000 
                 45343000 
                 46677400 
                 47985500 
               
               
                 29484500 
                 35065700 
                 38060900 
                 42419200 
                 44173600 
                 45364400 
                 46677800 
                 47985700 
               
               
                 30006300 
                 35142600 
                 38100300 
                 42442800 
                 44182200 
                 45373900 
                 46683700 
                 48024400 
               
               
                 30241900 
                 35169600 
                 38140400 
                 42543300 
                 44255200 
                 45396700 
                 46685300 
                 48041700 
               
               
                 30436300 
                 35201900 
                 38153300 
                 42546300 
                 44281900 
                 45431100 
                 46699900 
                 48048700 
               
               
                 30494600 
                 35203700 
                 38192600 
                 42551900 
                 44282200 
                 45446700 
                 46700100 
                 48054400 
               
               
                 30535200 
                 35217300 
                 38215600 
                 42595400 
                 44282300 
                 45448900 
                 46715700 
                 48070800 
               
               
                 30536400 
                 35227600 
                 38282500 
                 42625000 
                 44300500 
                 45470500 
                 46728100 
               
            
           
           
               
            
               
                 Tissue-specific hm-DLRs. Only 1000 selected DLRs are shown 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10421900 
                 16261100 
                 16954300 
                 17764700 
                 18920700 
                 23068700 
                 27303600 
                 27558300 
               
               
                 10589700 
                 16261600 
                 16956100 
                 17764900 
                 18921000 
                 23227200 
                 27303900 
                 27558700 
               
               
                 10596500 
                 16262900 
                 16956500 
                 17766800 
                 18923400 
                 23236000 
                 27304800 
                 27559900 
               
               
                 10596600 
                 16274900 
                 16962500 
                 17767800 
                 18926900 
                 23291400 
                 27306000 
                 27562900 
               
               
                 10598400 
                 16283000 
                 16975500 
                 17773300 
                 18931000 
                 23456600 
                 27307500 
                 27566800 
               
               
                 10598900 
                 16284300 
                 16976200 
                 17785700 
                 18943000 
                 23492500 
                 27307800 
                 27569500 
               
               
                 10715400 
                 16289300 
                 16976400 
                 17796400 
                 18952700 
                 23492900 
                 27307900 
                 27570000 
               
               
                 10736200 
                 16291100 
                 16989000 
                 17799200 
                 18956300 
                 23510900 
                 27308200 
                 27577000 
               
               
                 10843800 
                 16291200 
                 16989500 
                 17813700 
                 18956800 
                 23518700 
                 27316600 
                 27580900 
               
               
                 10913500 
                 16299300 
                 16990900 
                 17886800 
                 18968000 
                 23525700 
                 27324600 
                 27581400 
               
               
                 10924600 
                 16299800 
                 16991500 
                 17898600 
                 18970500 
                 23528800 
                 27326100 
                 27585800 
               
               
                 10955200 
                 16311900 
                 16992000 
                 17905500 
                 18971800 
                 23552200 
                 27327900 
                 27593300 
               
               
                 10987700 
                 16325200 
                 16997600 
                 17905800 
                 18973800 
                 23560200 
                 27330800 
                 27600300 
               
               
                 10992800 
                 16326500 
                 17001900 
                 17906900 
                 18975600 
                 23562800 
                 27335500 
                 27602200 
               
               
                 11012100 
                 16329200 
                 17009300 
                 17909300 
                 18977100 
                 23573000 
                 27335700 
                 27604900 
               
               
                 11028500 
                 16329400 
                 17017900 
                 17922600 
                 18977300 
                 23573400 
                 27337100 
                 27605000 
               
               
                 11094600 
                 16332100 
                 17034600 
                 17924100 
                 18982100 
                 23616700 
                 27338200 
                 27605300 
               
               
                 11098900 
                 16334600 
                 17036800 
                 17928200 
                 18987300 
                 23629800 
                 27340000 
                 27607200 
               
               
                 11112100 
                 16347100 
                 17040700 
                 17928300 
                 19005000 
                 23656900 
                 27340500 
                 27609200 
               
               
                 11122000 
                 16357400 
                 17041300 
                 17928400 
                 19010300 
                 23659900 
                 27341200 
                 27609700 
               
               
                 11130500 
                 16366000 
                 17041400 
                 17928900 
                 19020100 
                 23667300 
                 27342900 
                 27610200 
               
               
                 11131500 
                 16373300 
                 17041700 
                 17931600 
                 19023400 
                 23682600 
                 27343100 
                 27617500 
               
               
                 11132500 
                 16373500 
                 17045000 
                 17934400 
                 19028400 
                 23701700 
                 27343200 
                 27620800 
               
               
                 11139800 
                 16373700 
                 17045100 
                 17936200 
                 19031000 
                 23724200 
                 27351000 
                 27621100 
               
               
                 11144000 
                 16374700 
                 17046400 
                 17936400 
                 19033300 
                 23732400 
                 27352700 
                 27625200 
               
               
                 11144200 
                 16380800 
                 17049500 
                 17941100 
                 19033700 
                 23732500 
                 27354700 
                 27626300 
               
               
                 11145700 
                 16382700 
                 17050200 
                 17943400 
                 19033900 
                 23732700 
                 27360300 
                 27631000 
               
               
                 11170400 
                 16383100 
                 17065200 
                 17944100 
                 19034800 
                 23735900 
                 27360700 
                 27637700 
               
               
                 14384400 
                 16388400 
                 17080100 
                 17945100 
                 19035000 
                 23768500 
                 27362800 
                 27656300 
               
               
                 14804300 
                 16391900 
                 17080600 
                 17945800 
                 19041900 
                 23811300 
                 27363900 
                 27656400 
               
               
                 14816400 
                 16396200 
                 17084900 
                 17945900 
                 19042100 
                 23833900 
                 27369600 
                 27658700 
               
               
                 15056300 
                 16396800 
                 17085500 
                 17947600 
                 19045100 
                 23918100 
                 27371100 
                 27659000 
               
               
                 15067900 
                 16399900 
                 17089800 
                 17956700 
                 19045800 
                 23947700 
                 27372700 
                 27691600 
               
               
                 15068200 
                 16400700 
                 17094100 
                 17958000 
                 19046600 
                 23950600 
                 27373700 
                 27693400 
               
               
                 15077900 
                 16401600 
                 17099100 
                 17958400 
                 19048800 
                 24744100 
                 27375500 
                 27697600 
               
               
                 15166800 
                 16401700 
                 17099300 
                 17961700 
                 19052000 
                 24825100 
                 27375800 
                 27718300 
               
               
                 15227100 
                 16407000 
                 17116900 
                 17965900 
                 19052600 
                 24974800 
                 27376700 
                 27744600 
               
               
                 15228900 
                 16407900 
                 17117000 
                 17971500 
                 19063900 
                 25255800 
                 27377900 
                 27760700 
               
               
                 15261500 
                 16423400 
                 17121500 
                 17978700 
                 19070600 
                 25258400 
                 27381200 
                 27763400 
               
               
                 15261700 
                 16423900 
                 17123000 
                 17979900 
                 19071100 
                 25301000 
                 27382900 
                 27763500 
               
               
                 15262000 
                 16425600 
                 17127100 
                 18023400 
                 19077100 
                 25304000 
                 27383300 
                 27765300 
               
               
                 15297900 
                 16426600 
                 17142200 
                 18029900 
                 19098900 
                 25370100 
                 27384900 
                 27765800 
               
               
                 15300600 
                 16428100 
                 17145100 
                 18040100 
                 19100300 
                 25580300 
                 27387100 
                 27766600 
               
               
                 15309200 
                 16429800 
                 17147400 
                 18042800 
                 19101100 
                 25871200 
                 27388300 
                 27766800 
               
               
                 15357200 
                 16433200 
                 17153200 
                 18049200 
                 19102500 
                 26100000 
                 27389600 
                 27769700 
               
               
                 15375900 
                 16434800 
                 17154000 
                 18078000 
                 19104300 
                 26219400 
                 27397000 
                 27770900 
               
               
                 15380100 
                 16434900 
                 17156500 
                 18078200 
                 19104400 
                 26335300 
                 27397400 
                 27773000 
               
               
                 15381100 
                 16435500 
                 17157700 
                 18085600 
                 19108200 
                 26656500 
                 27399800 
                 27775500 
               
               
                 15383400 
                 16435600 
                 17166300 
                 18141500 
                 19108800 
                 26833400 
                 27407900 
                 27776200 
               
               
                 15383800 
                 16438600 
                 17172100 
                 18144200 
                 19116400 
                 26929000 
                 27410800 
                 27776400 
               
               
                 15384000 
                 16444100 
                 17174000 
                 18147500 
                 19116800 
                 26930500 
                 27411200 
                 27776700 
               
               
                 15384700 
                 16444300 
                 17176800 
                 18215200 
                 19117200 
                 26932600 
                 27411900 
                 27777100 
               
               
                 15386000 
                 16451600 
                 17178500 
                 18443200 
                 19117800 
                 26934200 
                 27414400 
                 27778500 
               
               
                 15386300 
                 16467200 
                 17180200 
                 18699900 
                 19119800 
                 26935800 
                 27417100 
                 27779300 
               
               
                 15404000 
                 16469800 
                 17182400 
                 18762800 
                 19128600 
                 26940700 
                 27417600 
                 27780400 
               
               
                 15407300 
                 16478900 
                 17182500 
                 18763400 
                 19131400 
                 26942300 
                 27424500 
                 27783600 
               
               
                 15412600 
                 16479500 
                 17187400 
                 18766800 
                 19136100 
                 26945900 
                 27428100 
                 27783800 
               
               
                 15431700 
                 16491000 
                 17188500 
                 18772200 
                 19150100 
                 26948800 
                 27428400 
                 27784100 
               
               
                 15434600 
                 16494700 
                 17193500 
                 18782400 
                 19151900 
                 26961400 
                 27430700 
                 27796700 
               
               
                 15435600 
                 16504800 
                 17193700 
                 18782600 
                 19162200 
                 26971700 
                 27431100 
                 27799400 
               
               
                 15436100 
                 16505800 
                 17197300 
                 18788400 
                 19166300 
                 26973100 
                 27431200 
                 27812100 
               
               
                 15436700 
                 16506400 
                 17206900 
                 18793000 
                 19167200 
                 26978700 
                 27434300 
                 27817200 
               
               
                 15436900 
                 16506500 
                 17207100 
                 18793300 
                 19167300 
                 26980800 
                 27440500 
                 27818000 
               
               
                 15442600 
                 16507400 
                 17210000 
                 18807500 
                 19173600 
                 26986400 
                 27440700 
                 27818500 
               
               
                 15442700 
                 16510800 
                 17211500 
                 18808900 
                 19174700 
                 26986600 
                 27443300 
                 27822300 
               
               
                 15443000 
                 16511000 
                 17212800 
                 18809700 
                 19175600 
                 26997800 
                 27443400 
                 27823900 
               
               
                 15443100 
                 16521700 
                 17213100 
                 18809900 
                 19177800 
                 26998000 
                 27445200 
                 27825700 
               
               
                 15444800 
                 16522600 
                 17218600 
                 18810300 
                 19188900 
                 26998200 
                 27446900 
                 27827800 
               
               
                 15447000 
                 16536900 
                 17221000 
                 18812500 
                 19196300 
                 27003800 
                 27448400 
                 27831100 
               
               
                 15448400 
                 16547800 
                 17222100 
                 18813900 
                 19196900 
                 27020000 
                 27449100 
                 27835300 
               
               
                 15451300 
                 16551900 
                 17226200 
                 18816600 
                 19202000 
                 27038000 
                 27449300 
                 27838000 
               
               
                 15452900 
                 16560600 
                 17232500 
                 18817500 
                 19213400 
                 27050200 
                 27450800 
                 27840200 
               
               
                 15453000 
                 16569600 
                 17236600 
                 18817600 
                 19228600 
                 27054700 
                 27452300 
                 27840900 
               
               
                 15455900 
                 16572400 
                 17247200 
                 18818700 
                 19278100 
                 27055500 
                 27459000 
                 27843400 
               
               
                 15456600 
                 16574500 
                 17247400 
                 18820500 
                 19280100 
                 27072600 
                 27463900 
                 27846500 
               
               
                 15457200 
                 16577000 
                 17261000 
                 18821800 
                 19294600 
                 27072900 
                 27465900 
                 27855900 
               
               
                 15458000 
                 16581900 
                 17268600 
                 18822500 
                 19311000 
                 27090600 
                 27467700 
                 27857900 
               
               
                 15464500 
                 16585300 
                 17277600 
                 18822900 
                 19318500 
                 27094500 
                 27468400 
                 27867400 
               
               
                 15464700 
                 16591800 
                 17279700 
                 18823100 
                 19345900 
                 27098000 
                 27468500 
                 27868200 
               
               
                 15465300 
                 16592400 
                 17280700 
                 18824500 
                 19514500 
                 27098200 
                 27469200 
                 27874100 
               
               
                 15468700 
                 16592500 
                 17300600 
                 18826000 
                 19764200 
                 27098400 
                 27470200 
                 27874900 
               
               
                 15471100 
                 16600400 
                 17305200 
                 18827900 
                 20037300 
                 27102000 
                 27471200 
                 27875500 
               
               
                 15473800 
                 16611500 
                 17305400 
                 18828300 
                 20173200 
                 27109900 
                 27476700 
                 27877200 
               
               
                 15474300 
                 16615800 
                 17333200 
                 18829500 
                 20216000 
                 27122500 
                 27477300 
                 27887800 
               
               
                 15474400 
                 16615900 
                 17341500 
                 18829800 
                 20250500 
                 27127200 
                 27479800 
                 27889100 
               
               
                 15477900 
                 16620100 
                 17350300 
                 18831300 
                 20270100 
                 27127300 
                 27481500 
                 27903600 
               
               
                 15491600 
                 16625100 
                 17352300 
                 18834100 
                 20508400 
                 27135600 
                 27485300 
                 27923600 
               
               
                 15491800 
                 16627700 
                 17353900 
                 18834500 
                 20649200 
                 27140300 
                 27489700 
                 27942700 
               
               
                 15552200 
                 16633100 
                 17354100 
                 18834600 
                 20966100 
                 27157500 
                 27490800 
                 27945000 
               
               
                 15647300 
                 16647900 
                 17356000 
                 18835600 
                 21390900 
                 27161300 
                 27491000 
                 27958500 
               
               
                 15650300 
                 16663300 
                 17357600 
                 18842600 
                 21540500 
                 27173500 
                 27493200 
                 27960200 
               
               
                 15705500 
                 16664500 
                 17362400 
                 18843000 
                 21546100 
                 27184800 
                 27495100 
                 27960300 
               
               
                 15731300 
                 16670800 
                 17363500 
                 18848200 
                 21594000 
                 27185000 
                 27497800 
                 27963700 
               
               
                 15734600 
                 16672300 
                 17371300 
                 18849100 
                 22347600 
                 27185100 
                 27498300 
                 28021600 
               
               
                 15743100 
                 16673600 
                 17376500 
                 18853800 
                 22367200 
                 27190500 
                 27500200 
                 28026000 
               
               
                 15748000 
                 16677500 
                 17377500 
                 18854600 
                 22369700 
                 27192700 
                 27502400 
                 28027800 
               
               
                 15748600 
                 16688000 
                 17422500 
                 18858800 
                 22370000 
                 27194600 
                 27503000 
                 28031600 
               
               
                 15758400 
                 16710100 
                 17433700 
                 18861900 
                 22370200 
                 27200700 
                 27503200 
                 28041800 
               
               
                 15765100 
                 16717600 
                 17442900 
                 18862400 
                 22381400 
                 27207100 
                 27504200 
                 28047900 
               
               
                 15807300 
                 16729800 
                 17443400 
                 18864000 
                 22386400 
                 27207500 
                 27505400 
                 28049900 
               
               
                 15811600 
                 16732800 
                 17457400 
                 18865600 
                 22396900 
                 27208700 
                 27508300 
                 28051800 
               
               
                 15851300 
                 16769900 
                 17485500 
                 18866400 
                 22397500 
                 27213300 
                 27509100 
                 28056300 
               
               
                 15854600 
                 16799800 
                 17485700 
                 18867100 
                 22399400 
                 27214200 
                 27509200 
                 28074300 
               
               
                 15869800 
                 16815100 
                 17489400 
                 18867400 
                 22413800 
                 27217000 
                 27510800 
                 28075700 
               
               
                 15983000 
                 16816700 
                 17496000 
                 18868000 
                 22421500 
                 27218200 
                 27511400 
                 28080900 
               
               
                 16007100 
                 16818200 
                 17505000 
                 18868600 
                 22429800 
                 27232100 
                 27511500 
                 28081500 
               
               
                 16016200 
                 16828600 
                 17541600 
                 18870800 
                 22440800 
                 27243600 
                 27518500 
                 28081900 
               
               
                 16105700 
                 16842900 
                 17542100 
                 18872900 
                 22452600 
                 27247400 
                 27518800 
                 28093200 
               
               
                 16197300 
                 16854200 
                 17544000 
                 18873100 
                 22461200 
                 27252100 
                 27519700 
                 28094900 
               
               
                 16202200 
                 16855700 
                 17552100 
                 18876400 
                 22493100 
                 27256300 
                 27522300 
                 28095900 
               
               
                 16213600 
                 16859000 
                 17552200 
                 18878800 
                 22514900 
                 27258500 
                 27522600 
                 28100300 
               
               
                 16213800 
                 16866200 
                 17565400 
                 18879100 
                 22537800 
                 27260300 
                 27524100 
                 28104200 
               
               
                 16213900 
                 16867200 
                 17568100 
                 18880800 
                 22537900 
                 27261700 
                 27524400 
                 28105700 
               
               
                 16214600 
                 16872200 
                 17568500 
                 18883100 
                 22555600 
                 27275300 
                 27527500 
                 28105800 
               
               
                 16215100 
                 16872400 
                 17612000 
                 18891400 
                 22564300 
                 27276400 
                 27528200 
                 28106000 
               
               
                 16222100 
                 16893900 
                 17636600 
                 18894500 
                 22572100 
                 27277200 
                 27529400 
                 28106900 
               
               
                 16240400 
                 16894500 
                 17651900 
                 18894600 
                 22573400 
                 27278600 
                 27529900 
                 28107200 
               
               
                 16241200 
                 16914400 
                 17653200 
                 18894900 
                 22591200 
                 27281000 
                 27534800 
                 28107900 
               
               
                 16248200 
                 16915700 
                 17659500 
                 18895500 
                 22619600 
                 27281400 
                 27537800 
                 28108900 
               
               
                 16248900 
                 16916300 
                 17675200 
                 18901000 
                 22620800 
                 27281900 
                 27537900 
                 28109400 
               
               
                 16249300 
                 16932100 
                 17675400 
                 18902800 
                 22631200 
                 27282500 
                 27538800 
                 28109800 
               
               
                 16251500 
                 16936900 
                 17690400 
                 18904100 
                 22640300 
                 27298500 
                 27544700 
                 28110400 
               
               
                 16253600 
                 16940600 
                 17728700 
                 18908700 
                 22651800 
                 27298600 
                 27551800 
                 28112300 
               
               
                 16255000 
                 16948100 
                 17741300 
                 18912400 
                 22728200 
                 27300300 
                 27552500 
                 28114800 
               
               
                 16260700 
                 16952900 
                 17763100 
                 18913800 
                 22737400 
                 27303000 
                 27553700 
                 28114900 
               
            
           
           
               
            
               
                 The selected T21-specific hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 27300500 
                 35948700 
                 39631800 
                 43336700 
                 45754400 
                 48054800 
                 40036100 
                 45715400 
               
               
                 27447600 
                 36053400 
                 39790900 
                 43722500 
                 46170300 
                 15496700 
                 40305700 
                 45747100 
               
               
                 30341400 
                 36175700 
                 39841200 
                 43763200 
                 46261400 
                 15841200 
                 40411000 
                 45884900 
               
               
                 30692000 
                 36185500 
                 40204700 
                 43896900 
                 46387900 
                 16481500 
                 42682100 
                 46235600 
               
               
                 32936900 
                 36215200 
                 40303500 
                 44427300 
                 46551600 
                 20885400 
                 43256500 
                 46463000 
               
               
                 32942700 
                 36381000 
                 40340900 
                 44511200 
                 46984700 
                 34790500 
                 43319100 
                 46851000 
               
               
                 33019400 
                 37847300 
                 40704800 
                 44615300 
                 47183600 
                 35616700 
                 43418800 
                 46932200 
               
               
                 33801400 
                 38262700 
                 40717600 
                 44906600 
                 47707300 
                 35894400 
                 43932600 
                 47983300 
               
               
                 34419100 
                 38327400 
                 40973900 
                 44916000 
                 47844900 
                 35913900 
                 44775800 
               
               
                 35203200 
                 38434500 
                 42694700 
                 45546000 
                 47897800 
                 35936600 
                 45244300 
               
               
                 35937700 
                 39484900 
                 43127700 
                 45753000 
                 47947900 
                 35948600 
                 45331400 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Selected u-CG-DLRs and hm-CG-DLRs for  
               
               
                 fetal T21 and fetal gender determination 
               
            
           
           
               
               
               
            
               
                   
                 DLR type 
                 Position 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 29732020 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 33462648 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 34672959 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 36193512 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 40801830 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 44303692 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 44741616 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 uCG 
                 chr21 45798427 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 30341466 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 35898716 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 38327475 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 40074274 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 40135661 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 44084933 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 45546038 
               
               
                   
                 Detection of fetal T21 aneuploidy 
                 hmCG 
                 chr21 46964859 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 22425661 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 50774868 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 23776534 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 9624546 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 9389347 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 62584036 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrX 138802442 
               
               
                   
                 Fetal gender determination 
                 uCG 
                 chrY 14774154 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 [The list of selected DLRs in chromosome 13 and 18. The coordinate 
               
               
                 is shown for the first base pair of 100 bp u-DLRs and hm-DLRs] 
               
               
                   
               
             
            
               
                 Pregnancy-specific chr13 u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 100008100 
                 101482600 
                 104426200 
                 108936500 
                 110709500 
                 112672700 
                 113632800 
                 113742100 
               
               
                 100038800 
                 101710500 
                 104949900 
                 109038400 
                 110846700 
                 112681700 
                 113649800 
                 113761500 
               
               
                 100066600 
                 101742200 
                 105608400 
                 109386000 
                 111057200 
                 112690800 
                 113653700 
                 114185600 
               
               
                 100315300 
                 101779900 
                 106272200 
                 109429500 
                 111090000 
                 113103000 
                 113673300 
                 114187400 
               
               
                 100392100 
                 101961000 
                 106323700 
                 109819300 
                 111773200 
                 113138200 
                 113684400 
                 114203600 
               
               
                 100479400 
                 102346200 
                 106590100 
                 109944500 
                 111852400 
                 113279700 
                 113694800 
                 114215200 
               
               
                 100529300 
                 102578800 
                 106662300 
                 109949500 
                 111997000 
                 113416500 
                 113697000 
                 114441300 
               
               
                 100570600 
                 102811900 
                 107601600 
                 110174500 
                 112101500 
                 113420400 
                 113698300 
                 114458800 
               
               
                 100575700 
                 102906700 
                 108033300 
                 110178400 
                 112226000 
                 113532000 
                 113707900 
                 114471100 
               
               
                 100596500 
                 103155900 
                 108233300 
                 110193200 
                 112288900 
                 113544500 
                 113709900 
               
               
                 101100600 
                 103702300 
                 108310100 
                 110254600 
                 112293800 
                 113551300 
                 113715800 
               
               
                 101185900 
                 103951500 
                 108413600 
                 110481200 
                 112623000 
                 113551800 
                 113731600 
               
               
                 101313000 
                 104351000 
                 108869700 
                 110653400 
                 112664300 
                 113556700 
                 113739100 
               
            
           
           
               
            
               
                 Pregnancy-specific chr18 u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10164100 
                 11127100 
                 1225900 
                 13326200 
                 14966900 
                 21431200 
                 23230100 
                 28368600 
               
               
                 10230000 
                 11280000 
                 12431600 
                 13421100 
                 14970000 
                 21579300 
                 23449800 
                 29048800 
               
               
                 10248300 
                 11283300 
                 12561500 
                 13431900 
                 18700600 
                 21587400 
                 24037600 
                 29144400 
               
               
                 10263100 
                 11378600 
                 12565400 
                 13432000 
                 19028000 
                 21668700 
                 24125900 
                 29926800 
               
               
                 10272700 
                 11378800 
                 12723100 
                 13497800 
                 1911400 
                 21709600 
                 24318400 
                 30488500 
               
               
                 10433300 
                 11532400 
                 12741900 
                 13511300 
                 19222900 
                 21972200 
                 24360100 
                 30722200 
               
               
                 10563500 
                 11750800 
                 13135100 
                 13517000 
                 19273800 
                 22278700 
                 24421300 
                 31581500 
               
               
                 10706400 
                 11759500 
                 13226200 
                 13527500 
                 19294100 
                 22307200 
                 24459600 
                 31941500 
               
               
                 10723700 
                 11802400 
                 13246300 
                 13625400 
                 19898200 
                 22733400 
                 24709600 
                 32154300 
               
               
                 10842000 
                 11817700 
                 13247600 
                 13627100 
                 19991500 
                 22783900 
                 24873100 
               
               
                 1091900 
                 11847900 
                 13254200 
                 13645500 
                 20008200 
                 22800300 
                 25465700 
               
               
                 10936900 
                 12035600 
                 13270000 
                 13647500 
                 20815500 
                 23006400 
                 25734400 
               
               
                 11101200 
                 12234800 
                 13278200 
                 14162800 
                 20895400 
                 23092300 
                 28207100 
               
            
           
           
               
            
               
                 Tissue-specific chr13 u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 111000000 
                 100286200 
                 100395800 
                 100591400 
                 101203000 
                 101763500 
                 102608900 
                 103229800 
               
               
                 100057600 
                 100291500 
                 100406500 
                 100596700 
                 101212600 
                 101820800 
                 102732300 
                 103236500 
               
               
                 100066900 
                 100315300 
                 100442900 
                 100656600 
                 101288800 
                 101825400 
                 102775200 
                 103354900 
               
               
                 100078400 
                 100318100 
                 100446000 
                 100689500 
                 101314300 
                 101885900 
                 102852000 
                 103365800 
               
               
                 100080400 
                 100328100 
                 100456000 
                 100704300 
                 101334100 
                 101931900 
                 102901900 
                 103400700 
               
               
                 100097800 
                 100341200 
                 100463200 
                 100932000 
                 101391500 
                 101996400 
                 102906700 
                 103427900 
               
               
                 100110300 
                 100344400 
                 100479400 
                 100989300 
                 101404700 
                 102271000 
                 102979800 
                 103430200 
               
               
                 100122700 
                 100358700 
                 100541600 
                 101034400 
                 101425600 
                 102293100 
                 103044900 
                 103539000 
               
               
                 100140900 
                 100375400 
                 100557000 
                 101045700 
                 101593600 
                 102397600 
                 103045200 
                 103547600 
               
               
                 100142900 
                 100375800 
                 100559900 
                 101097200 
                 101593900 
                 102498800 
                 103094700 
               
               
                 100152100 
                 100377300 
                 100563200 
                 101160600 
                 101596900 
                 102548800 
                 103174600 
               
               
                 100172600 
                 100387200 
                 100565000 
                 101194500 
                 101605400 
                 102558900 
                 103202900 
               
               
                 100271900 
                 100395500 
                 100570600 
                 101199900 
                 101742200 
                 102573800 
                 103207500 
               
            
           
           
               
            
               
                 Tissue-specific chr18 u-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10004000 
                 10218000 
                 10366100 
                 10472500 
                 10723700 
                 10825100 
                 10890600 
                 11037100 
               
               
                 10007600 
                 10218900 
                 10369400 
                 10472800 
                 10724200 
                 10828600 
                 10902200 
                 11049800 
               
               
                 10010500 
                 10220300 
                 10372500 
                 1047300 
                 10730800 
                 10834900 
                 10925700 
                 11054500 
               
               
                 10013200 
                 10230100 
                 10373000 
                 10500900 
                 10731400 
                 10838000 
                 10936900 
                 11058000 
               
               
                 10029700 
                 10231800 
                 10381300 
                 10571200 
                 10734700 
                 10838800 
                 10971200 
                 11063500 
               
               
                 10030300 
                 10263000 
                 10398500 
                 10582400 
                 10737400 
                 10844200 
                 10986100 
                 11068000 
               
               
                 10035100 
                 10272700 
                 10403400 
                 10592900 
                 10745300 
                 10844600 
                 10990400 
                 11071000 
               
               
                 10052400 
                 10287000 
                 10404600 
                 10682200 
                 10755600 
                 10845500 
                 10993600 
                 11074000 
               
               
                 10068500 
                 10300900 
                 10408400 
                 10682500 
                 10774400 
                 10847900 
                 10994000 
                 11099500 
               
               
                 10100200 
                 10301900 
                 10410400 
                 10703600 
                 10775500 
                 10864900 
                 11009700 
               
               
                 10120900 
                 10332200 
                 10423100 
                 10708300 
                 10776700 
                 10873300 
                 11012000 
               
               
                 10153400 
                 10343100 
                 10433300 
                 10717300 
                 10785400 
                 10878300 
                 11021400 
               
               
                 10164600 
                 10346600 
                 10450400 
                 10721900 
                 10806500 
                 10888800 
                 11027200 
               
            
           
           
               
            
               
                 Pregnancy-specific chr13 hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 100305000 
                 107282100 
                 114215200 
                 28102000 
                 43394700 
                 46000500 
                 50682600 
                 92422900 
               
               
                 100776700 
                 110322500 
                 20563600 
                 28571600 
                 44470900 
                 47112300 
                 50238200 
                 99310800 
               
               
                 107214300 
                 111140600 
                 21527500 
                 28936500 
                 45023500 
               
            
           
           
               
            
               
                 Pregnancy-specific chr18 hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 24079700 
                 3473100 
                 56528700 
                 72186200 
                 9478700 
                 3172300 
                 35234800 
                 61869500 
               
               
                 73934900 
               
            
           
           
               
            
               
                 Tissue-specific chr13 hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 111000000 
                 100756100 
                 101237300 
                 101687300 
                 101833600 
                 102068100 
                 102116200 
                 103304300 
               
               
                 100015300 
                 100765100 
                 101242700 
                 101701700 
                 101837400 
                 102076800 
                 102168800 
                 103304500 
               
               
                 100033700 
                 100828300 
                 101255600 
                 101702000 
                 101898100 
                 102078200 
                 102183900 
                 103344400 
               
               
                 100078000 
                 100931800 
                 101263700 
                 101710300 
                 101956100 
                 102082400 
                 102204700 
                 103349700 
               
               
                 100084900 
                 100980400 
                 101281000 
                 101734400 
                 101960500 
                 102102200 
                 102206000 
                 103358800 
               
               
                 100085400 
                 100982800 
                 101286200 
                 101751100 
                 101961200 
                 102105900 
                 102228800 
                 103362900 
               
               
                 100126100 
                 101075300 
                 101305600 
                 101751500 
                 101990200 
                 102106400 
                 102238000 
                 103408800 
               
               
                 100136900 
                 101094300 
                 101320900 
                 101764100 
                 101991600 
                 102106800 
                 102344000 
                 103481700 
               
               
                 100138400 
                 101098400 
                 101365300 
                 101777000 
                 101992300 
                 102108000 
                 102407700 
                 105737500 
               
               
                 100211300 
                 101122000 
                 101399900 
                 101794700 
                 102007400 
                 102108700 
                 102553800 
               
               
                 100231000 
                 101182400 
                 101451600 
                 101796000 
                 102052700 
                 102109100 
                 102580000 
               
               
                 100243900 
                 101199800 
                 101525100 
                 101799300 
                 102060500 
                 102109800 
                 103259500 
               
               
                 100589400 
                 101202400 
                 101533600 
                 101831100 
                 102060600 
                 102112500 
                 103265400 
               
            
           
           
               
            
               
                 Tissue-specific chr18 hm-DLRs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10018900 
                 10207900 
                 10935800 
                 11956100 
                 12326400 
                 12659400 
                 12855600 
                 13622800 
               
               
                 10020300 
                 10373100 
                 11208700 
                 11971200 
                 12367700 
                 12660600 
                 12871700 
                 1379600 
               
               
                 10030100 
                 10377900 
                 11274300 
                 12027100 
                 12375000 
                 12734300 
                 12908700 
                 1399600 
               
               
                 10034200 
                 10547100 
                 11571800 
                 12027200 
                 12389700 
                 12738100 
                 12908900 
                 14090000 
               
               
                 10045400 
                 10560700 
                 11690300 
                 12231400 
                 12443300 
                 12738600 
                 1296400 
                 14975600 
               
               
                 10046100 
                 10710600 
                 11807300 
                 12251400 
                 12463100 
                 12738700 
                 12969400 
                 15021300 
               
               
                 10052400 
                 107600 
                 11829700 
                 12254300 
                 12467300 
                 12748100 
                 12972900 
                 18573300 
               
               
                 10055600 
                 10796000 
                 11857200 
                 12254900 
                 12476900 
                 12775300 
                 12995300 
                 18637700 
               
               
                 10073000 
                 10798100 
                 11900 
                 12255500 
                 12521400 
                 12782800 
                 12996400 
                 18710400 
               
               
                 10093500 
                 10799200 
                 11912400 
                 12282900 
                 12547600 
                 12788800 
                 13137700 
               
               
                 10121200 
                 10923400 
                 11921600 
                 12289600 
                 12565700 
                 12839200 
                 1331300 
               
               
                 10169200 
                 10927300 
                 11947700 
                 12301800 
                 12641500 
                 12849500 
                 13608200 
               
               
                 10190600 
                 10929600 
                 11952800 
                 12324700 
                 12646700 
                 12850200 
                 13611300 
               
               
                   
               
            
           
         
       
     
     NON PATENT LITERATURE 
     
         
         NPL1: Akolekar R, Beta J, Picciarelli G, Ogilvie C, D&#39;Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2015 January; 45(1):16-26. doi: 10.1002/uog.14636. 
         NPL2: Chan K C, Zhang J, Hui A B, Wong N, Lau T K, Leung T N, Lo K W, Huang D W, Lo Y M. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. 2004 January; 50(1):88-92. 
         NPL3: Chim S S, Jin S, Lee T Y, Lun F M, Lee W S, Chan L Y, Jin Y, Yang N, Tong Y K, Leung T Y, Lau T K, Ding C, Chiu R W, Lo Y M. Systematic search for placental DNA-methylation markers on chromosome 21: toward a maternal plasma-based epigenetic test for fetal trisomy 21. Clin Chem. 2008 March; 54(3):500-11. 
         NPL3: Chim S S, Tong Y K, Chiu R W, Lau T K, Leung T N, Chan L Y, Oudejans C B, Ding C, Lo Y M. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci USA. 2005 Oct. 11; 102(41):14753-8. 
         NPL4: Chiu R W, Chan K C, Gao Y, Lau V Y, Zheng W, Leung T Y, Foo C H, Xie B, Tsui N B, Lun F M, Zee B C, Lau T K, Cantor C R, Lo Y M. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Version 2. Proc Natl Acad Sci USA. 2008 Dec. 23; 105(51):20458-63. 
         NPL5: Daniels G, Finning K, Martin P, Summers J. Fetal blood group genotyping: present and future. Ann N Y Acad Sci. 2006 September; 1075:88-95. 
         NPL6: Fan H C, Blumenfeld Y J, Chitkara U, Hudgins L, Quake S R. Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Clin Chem. 2010 August; 56(8):1279-86. 
         NPL7: Gibas P, Narmontė M, Staševskij Z, Gordevičius J, Klimašauskas S, Kriukienė E. Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing. PLoS Biol. 2020 accepted 
         NPL8: Jensen T J, Kim S K, Zhu Z, Chin C, Gebhard C, Lu T, Deciu C, van den Boom D, Ehrich M. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains. Genome Biol. 2015 Apr. 15; 16(1):78. 
         NPL9: Keravnou A, Ioannides M, Tsangaras K, Loizides C, Hadjidaniel M D, Papageorgiou E A, Kyriakou S, Antoniou P, Mina P, Achilleos A, Neofytou M, Kypri E, Sismani C, Koumbaris G, Patsalis P C. Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions. Genet Res (Camb). 2016 Nov. 11; 98:e15. 
         NPL10: Kriukienė E, Labrie V, Khare T, Urbanavičiūtė G, Lapinaitė A, Koncevičius K, Li D, Wang T, Pai S, Ptak C, Gordevičius J, Wang S C, Petronis A, Klimašauskas S. DNA unmethylome profiling by covalent capture of CpG sites. Nat Commun. 2013; 4:2190. 
         NPL11: Li Y, Zimmermann B, Rusterholz C, Kang A, Holzgreve W, Hahn S. Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin Chem 2004; 50: 1002-11. 
         NPL12: Lo Y M, Chan K C, Sun H, Chen E Z, Jiang P, Lun F M, Zheng Y W, Leung T Y, Lau T K, Cantor C R, Chiu R W. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. 2010 Dec. 8; 2(61):61ra91. 
         NPL13: Lo Y M, Chiu R W. Prenatal diagnosis: progress through plasma nucleic acids. Nat Rev Genet. 2007 January; 8(1):71-7. 
         NPL14: Lo Y M, Corbetta N, Chamberlain P F, Rai V, Sargent I L, Redman C W, Wainscoat J S. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350:485-487. 
         NPL15: Lo Y M, Hjelm N M, Fidler C, Sargent I L, Murphy M F, Chamberlain P F, Poon P M, Redman C W, Wainscoat J S. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med. 1998 Dec. 10; 339(24):1734-8. 
         NPL16: Lun F M, Chiu R W, Sun K, Leung T Y, Jiang P, Chan K C, Sun H, Lo Y M. Noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of maternal plasma DNA. Clin Chem. 2013 November; 59(11):1583-94. 
         NPL17: Masevičius V, Nainytė M, Klimašauskas S. Synthesis of S-Adenosyl-L-Methionine Analogs with Extended Transferable Groups for Methyltransferase-Directed Labeling of DNA and RNA. Curr Protoc Nucleic Acid Chem. 2016 Mar. 1; 64:1.36.1-1.36.13. 
         NPL18: Old R W, Crea F, Puszyk W, Hulten M A. Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome. Reprod Biomed Online. 2007 August; 15(2):227-35. 
         NPL19: Papageorgiou E A, Fiegler H, Rakyan V, Beck S, Hulten M, Lamnissou K, Carter N P, Patsalis P C. Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies. Am J Pathol. 2009 May; 174(5):1609-18. 
         NPL20: Parker S E, Mai C T, Canfield M A, Rickard R, Wang Y, Meyer R E, Anderson P, Mason C A, Collins J S, Kirby R S, Correa A; National Birth Defects Prevention Network. Updated National Birth Prevalence estimates for selected birth defects in the United States, 2004-2006. Birth Defects Res A Clin Mol Teratol. 2010 December; 88(12):1008-16. 
         NPL21: Song C X, Szulwach K E, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen C H, Zhang W, Jian X, Wang J, Zhang L, Looney T J, Zhang B, Godley L A, Hicks L M, Lahn B T, Jin P, He C. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol. 2011 January; 29(1):68-72. 
         NPL22: Staševskij Z, Gibas P, Gordevičius J, Kriukienė E, Klimašauskas S. Tethered Oligonucleotide-Primed Sequencing, TOP-Seq: A High-Resolution Economical Approach for DNA Epigenome Profiling. Mol Cell. 2017 Feb. 2; 65(3):554-564.e6. 
         NPL23: Tong Y K, Jin S, Chiu R W, Ding C, Chan K C, Leung T Y, Yu L, Lau T K, Lo Y M. Noninvasive prenatal detection of trisomy 21 by an epigenetic-genetic chromosome-dosage approach. Clin Chem. 2010 January; 56(1):90-8. 
         NPL24: Tsaliki E, Papageorgiou E A, Spyrou C, Koumbaris G, Kypri E, Kyriakou S, Sotiriou C, Touvana E, Keravnou A, Karagrigoriou A, Lamnissou K, Velissariou V, Patsalis P C. MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21. Prenat Diagn. 2012 October; 32(10):996-1001. 
         NPL25: Weber M, Davies J J, Wittig D, Oakeley E J, Haase M, Lam W L, Schübeler D. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 2005 August; 37(8):853-62. 
         NPL26: Zimmermann B, Holzgreve W, Wenzel F, Hahn S. Novel real-time quantitative PCR test for trisomy 21. Clin Chem. 2002 February; 48(2):362-3.