U.S. patent application number 16/538231 was filed with the patent office on 2020-07-02 for systems for detecting dna orginating from different individuals.
The applicant listed for this patent is The Chinese University of Hong Kong. Invention is credited to YUK MING Dennis LO, Lit Man Poon.
Application Number | 20200208225 16/538231 |
Document ID | / |
Family ID | 25482355 |
Filed Date | 2020-07-02 |
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United States Patent
Application |
20200208225 |
Kind Code |
A1 |
LO; YUK MING Dennis ; et
al. |
July 2, 2020 |
SYSTEMS FOR DETECTING DNA ORGINATING FROM DIFFERENT INDIVIDUALS
Abstract
In a first aspect, the present invention features methods for
differentiating DNA species originating from different individuals
in a biological sample. These methods may be used to differentiate
or detect fetal DNA in a maternal sample or to differentiate DNA of
an organ donor from DNA of an organ recipient. In preferred
embodiments, the DNA species are differentiated by observing
epigenetic differences in the DNA species such as differences in
DNA methylation. In a second aspect, the present invention features
methods of detecting genetic abnormalities in a fetus by detecting
fetal DNA in a biological sample obtained from a mother. In a third
aspect, the present invention features methods for differentiating
DNA species originating from an organ donor from those of an organ
recipient. In a fourth aspect, the present invention features kits
for differentiating DNA species originating from different
individuals in a biological sample.
Inventors: |
LO; YUK MING Dennis;
(Homantin, CN) ; Poon; Lit Man; (Tsuen Wan,
HK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Chinese University of Hong Kong |
Hong Kong |
|
CN |
|
|
Family ID: |
25482355 |
Appl. No.: |
16/538231 |
Filed: |
August 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14599082 |
Jan 16, 2015 |
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16538231 |
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13855974 |
Apr 3, 2013 |
8962280 |
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14599082 |
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13437584 |
Apr 2, 2012 |
8431343 |
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13855974 |
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12250480 |
Oct 13, 2008 |
8168382 |
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13437584 |
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11075092 |
Mar 7, 2005 |
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12250480 |
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09944951 |
Aug 31, 2001 |
6927028 |
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11075092 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 2600/154 20130101; C12Q 1/6888 20130101; C12Q 1/6876
20130101 |
International
Class: |
C12Q 1/6888 20060101
C12Q001/6888; C12Q 1/6876 20060101 C12Q001/6876 |
Claims
1-38. (canceled)
39. A method for determining methylation status of a human DNA
species that is present outside of cells in a biological sample
obtained from a human, wherein the sample (1) contains human DNA
that is originated from two different human individuals and present
outside of cells; and (2) is obtained from one of the two different
human individuals, the method comprising the steps of: (a) treating
the sample with a reagent that differentially reacts with
methylated and unmethylated DNA; and (b) detecting the presence of
a methylated version and/or an unmethylated version of the human
DNA species in the sample, thereby determining methylation status
of the human DNA species in the sample.
40. The method of claim 39, further comprising measuring
concentration of the methylated or unmethylated version of the
human DNA species.
41. The method of claim 39, further comprising purifying the
methylated or unmethylated version of the human DNA species.
42. The method of claim 39, wherein the reagent is sodium
bisulfite.
43. The method of claim 39, wherein step (b) comprises an
amplification reaction.
44. The method of claim 43, wherein the amplification reaction is a
polymerase chain reaction (PCR).
45. The method of claim 44, wherein the PCR is a
methylation-specific PCR.
46. The method of claim 39, wherein step (b) comprises DNA
sequencing.
47. The method of claim 39, wherein step (b) comprises a primer
extension assay.
48. The method of claim 39, wherein the sample is a fluid or
cellular sample or a mixture thereof.
49. The method of claim 39, wherein the sample is a blood
sample.
50. The method of claim 39, wherein the sample is a serum or plasma
sample.
51. The method of claim 39, wherein the two different human
individuals are a pregnant woman and a fetus the woman is
carrying.
52. The method of claim 51, wherein the sample is a maternal blood
sample.
53. The method of claim 51, wherein the sample is a maternal serum
or plasma sample.
54. The method of claim 39, wherein the different human individuals
are a transplant recipient and a transplant donor.
55. The method of claim 54, wherein the transplant is a bone marrow
transplant.
56. The method of claim 39, wherein the two different human
individuals are one male and one female.
57. The method of claim 39, wherein step (b) is performed inside a
cell.
58. The method of claim 57, wherein step (b) comprises an in situ
methylation-specific PCR.
59. The method of claim 57, further comprising isolation of cells
based on methylation difference of the DNA species.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/599,082, filed Jan. 16, 2015, which is a continuation of
U.S. application Ser. No. 13/855,974, filed Apr. 3, 2013, which is
a continuation of U.S. application Ser. No. 13/437,584, filed Apr.
2, 2012, now U.S. Pat. No. 8,431,343, which is a continuation of
U.S. application Ser. No. 12/250,480, filed Oct. 13, 2008, now U.S.
Pat. No. 8,168,382, which is a continuation of U.S. application
Ser. No. 11/075,092, filed Mar. 7, 2005, now abandoned, which is a
divisional application of U.S. application Ser. No. 09/944,951,
filed Aug. 31, 2001, now U.S. Pat. No. 6,927,028, the contents of
all of the above are incorporated herein in the entirety by
reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] The presence of DNA originating from different individuals
in bodily fluids is a well-known biological phenomenon in many
clinical and biological scenarios. For example, following bone
marrow transplantation, the hemopoietic system of the
transplantation recipient will consist of varying proportions of
donor's and recipient's cells. The ascertainment of the amount of
donor's or recipient's cells has been performed by the detection of
genetic differences between the donor and recipient, including
gender (Mangioni et al., Bone Marrow Transplant 20:969-73 (1997))
and DNA polymorphisms (Roux et al., Blood 79:2775-83 (1992)). The
corollary of this approach is that if the analysed region does not
bear a genetic difference between the donor and recipient, then
analysis by the current approach will not be possible.
[0003] In another example, during pregnancy, detection of fetal DNA
in maternal plasma and serum has been previously demonstrated (Lo
et al., Lancet 350:9076: 485-7 (1997)). This technology has
demonstrated that fetal DNA isolated from maternal plasma and serum
can be used for non-invasive prenatal diagnosis (Lo et al., N Eng J
Med, 339 (24):1734-8 (1998); Faas et al., Lancet 352 (9135):1196
(1998); Amicucci et al., Clin Chem 46 (2):301 (2000); Chen et al.,
Prenat Dia 20 (4):355-7 (2000); Saito et al., Lancet 356:1170
(2000)). The clinical application of this phenomenon has been
helped by the relatively high absolute and relative concentrations
of such circulating fetal DNA in maternal plasma and serum (Lo et
al., Am J. Hum Genet 62:768-775 (1998)). Using this approach,
noninvasive prenatal detection of a number of conditions has been
achieved, including fetal rhesus D status (Lo et al., New Eng J Med
339:1734-1738 (1998)), myotonic dystrophy (Amicucci et al., Clin
Chem 46:301-302 (2000)), achondroplasia (Saito et al., Lancet
356:1170 (2000)) and certain chromosomal translocations (Chen et
al., Prenat Diag 20:335-357 (2000); Chen et al., Clin Chem
47:937-939 (2001)). All of these current approaches have utilized
the detection of DNA sequences inherited from the father and which
are genetically distinguishable from those of the mother (Bianchi,
Am J Hum Genet 62 (4):763 (1998). Specifically, the detection of
DNA that the fetus has inherited from the mother in maternal plasma
or serum has been thought to be impossible. Similar limitations
have also been described for the detection of fetal nucleated cells
isolated from the cellular fraction of maternal blood (Lo et al.,
Ann N Y Acad Sci, 731:204 (1994).
[0004] Others have detected aberrantly methylated DNA from cancer
patients. This has been reported for patients with a variety of
cancers, including lung (Esteller, et al., Cancer Res 59 (1):67
(1999)) and liver cancer (Wong et al., Cancer Res 59 (1):71
(1999)).
[0005] Recently, much interest has been focused on the biology of
epigenetic phenomena, namely processes which alter the phenotype
but which are not associated with changes in DNA sequence (Wolffe,
Science 286:481-486 (1999)). One of the best characterised
epigenetic processes is DNA methylation (Wolffe et al., Curr Biol.
10:R463-R465 (1999)). A method for discriminating DNA species
originating from different individuals in biological fluids using
epigenetic, rather than genetic differences between the DNA species
would be highly valuable. For example, the epigenetic detection of
fetal DNA in a maternal sample would provide a significant
advancement enabling additional screening and diagnostic
methods.
SUMMARY OF THE INVENTION
[0006] In a first aspect, the present invention features methods
for differentiating DNA species originating from different
individuals in a biological sample. In preferred embodiments the
methods of the present invention are used to differentiate or
detect fetal DNA in a maternal sample or to differentiate DNA of an
organ donor from DNA of an organ recipient.
[0007] Those of skill in the art will appreciate that the
biological sample obtained from an individual may be taken from any
fluid or cell sample, however, in preferred embodiments the bodily
fluid is plasma or serum. In preferred embodiments, the DNA species
are differentiated by observing epigenetic differences in the DNA
species such as differences in DNA methylation. For instance, in
situations where one DNA species comes from a male, and one DNA
species comes from a female, the epigenetic marker may be the
inactivated X chromosome of the female individual. In such
embodiments, methylated DNA sequences on the inactivated X
chromosome may be used to detect DNA originating from the female
individual. In some embodiments, the epigenetic differences may be
analyzed inside cells. Further, in some embodiments, the epigenetic
differences may be analyzed using in-situ methylation-specific
polymerase chain reaction. Additionally, the epigenetic differences
may be used to sort or isolate cells from the respective
individuals or to purify DNA from the respective individuals. The
methods according to the present invention may be performed with or
without measuring the concentrations of DNA species, however, in
preferred embodiments, the concentrations of DNA species with the
respective epigenetic differences are measured. Such measuring of
concentrations involves measuring the respective DNA methylation
differences in embodiments wherein DNA methylation differences is
the epigenetic marker. In especially preferred embodiments, sodium
bisulfite is added to the biological sample or to the DNA species
directly to detect the DNA methylation differences. However, in
other embodiments a methylation-specific polymerase chain reaction,
as is well known to those skilled in the art, may be used to detect
the DNA methylation differences. In yet other embodiments, DNA
sequencing or primer extension may be used to detect the
methylation differences.
[0008] In a second aspect, the present invention features methods
of detecting abnormalities in a fetus by detecting fetal DNA in a
biological sample obtained from a mother. The methods according to
the present invention provide for detecting fetal DNA in a maternal
sample by differentiating the fetal DNA from the maternal DNA based
upon epigenetic markers such as differences in DNA methylation.
Employing such methods, fetal DNA that is predictive of a genetic
anomaly or genetically based disease may be identified thereby
providing methods for prenatal diagnosis. These methods are
applicable to any and all pregnancy-associated conditions for which
methylation changes associated with a disease state is identified.
Exemplary diseases that may be diagnosed include, for example,
preeclampsia, a chromosomal aneuploidy, including but not limited
to trisomy 21, Prader-Willi Syndrome, and Angelman Syndrome.
[0009] As with the broader differentiating methods of the first
aspect of the invention, the biological sample obtained from the
mother is preferably plasma or serum. The differentiation between
maternal and fetal DNA may be performed with or without quantifying
the concentration of fetal DNA in maternal plasma or serum. In
embodiments wherein the fetal DNA is quantified, the measured
concentration may be used to predict, monitor or diagnose or
prognosticate a pregnancy-associated disorder. In preferred
embodiments, the particular fetus-derived epigenetic mark is
associated with a fetal disorder, and in some embodiments an
epigenetic characteristic in fetal cells in the placenta is used as
a fetus-specific marker in maternal plasma or serum.
[0010] In a third aspect, the present invention features methods
for differentiating DNA species originating from an organ donor
from those of an organ recipient. As with the broader
differentiating methods of the first aspect of the invention, the
biological sample obtained is preferably plasma or serum. The
differentiation between DNA from the organ donor and organ
recipient or potential organ donor and potential organ recipient
may be performed with or without quantifying the concentration of
DNA in the biological sample. This embodiment is particularly
useful in instances when the transplantation is a bone marrow
transplantation. Such measurements may be used to predict the
clinical progress of the transplantation recipient especially as
regards organ rejection.
[0011] In a fourth aspect, the present invention features kits for
differentiating DNA species originating from different individuals
in a biological sample. Such kits are useful, for instance, for
differentiating or detecting the presence of fetal DNA in a
maternal biological sample or for differentiating DNA from an organ
donor or potential organ donor from that of an organ recipient or
potential organ recipient. The kits according to the present
invention comprise one or more reagents for ascertaining the
methylation status of the maternal DNA such as sodium bisulfite and
one or more reagents for detecting the presence of DNA such as a
gel. Additionally, such kits may include one or more reagents for
amplifying the amount of DNA present in the sample such as one or
more reagents for performing polymerase chain reaction
amplification. Such reagents are well known to those of skill in
the art. Further, such kits may include one or more apparatuses for
obtaining a maternal DNA sample. Such apparatuses are well known to
those skilled in the art. In particular the kits according to the
present invention may be used for diagnosing a disease caused all
or in part by a genetic anomaly such as a mutation, substitution or
deletion in all or part of a DNA sequence present in a fetus.
Exemplary diseases that may be diagnosed include, for example,
preeclampsia, a chromosomal aneuploidy, including but not limited
to trisomy 21, Prader-Willi Syndrome and Angelman Syndrome.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 demonstrates the results of an assay detecting
methylated and unmethylated DNA sequences of the androgen receptor
gene. In total, 6 male and 11 female healthy subjects were
recruited. Of all male control subjects, only the unmethylated
androgen receptor gene was detected in these samples as expected
(FIG. 1A). By contrast, both unmethylated and methylated androgen
receptor gene DNA sequences were observed in female control
subjects (FIG. 1A). The detection rates of methylated and
unmethylated androgen receptor genes in these female subjects were
100% and 82%, respectively. When DNA samples were omitted from the
assay, no positive signal was observed (FIG. 1A). Interestingly,
positive signals for both methylated and unmethylated DNA sequences
were observed in all male bone marrow transplantation recipients
with female donors, indicating cells from female donor exist in the
blood circulation of male recipients.
[0013] FIG. 2 provides a schematic representation of the
differentially methylated region (DMR) of the human IGF2-H19
region. The two 450-bp repeat (A1 and A2) and seven 400-bp repeat
(B1-B7) units are shown. The potential methylation sites on the
upper strand DNA of the studied region are represented by open
circles. The studied single nucleotide polymorphism (SNP) site
(A/G) is indicated by an open box. Open arrows represent the
location of the forward (for) and reverse (rev) primers in PCR
reactions specific for the methylated (M) and unmethylated (U)
alleles, respectively. Sequences of these MSP primers are shown.
Sequence differences between bisulfite-treated DNA and untreated
DNA are highlighted in bold italics and sequence differences
between methylated (paternally-inherited) and unmethylated
(maternally-inherited) DNA are underlined in bold.
[0014] FIG. 3 demonstrates detection of methylated
(paternally-inherited) fetal DNA in 3.sup.rd trimester (a) and
2.sup.nd trimester (b) maternal plasma. DNA sequence of methylated
alleles in maternal buffy coat (panel 1), fetal buffy coat or
amniotic fluid (panel 2), prenatal maternal plasma (panel 3) and
postnatal maternal plasma (panel 4) samples are shown. The presence
of methylated fetal DNA in the prenatal maternal plasma sample is
indicated by *. The polymorphic (SNP) site is shown in red
letters.
[0015] FIG. 4 demonstrates detection of unmethylated
(maternally-inherited) fetal DNA in maternal plasma. (a)
Unmethylated DNA sequences were detected in maternal buffy coat
(panel 1) and a third trimester maternal sample (panel 2) using
direct sequencing. The presence of unmethylated fetal DNA in
maternal plasma is indicated by *. (b) Unmethylated fetal DNA
(arrow) was detected in two third trimester maternal plasma samples
using the primer extension assay. (c) Unmethylated fetal DNA
(arrow) was detected in a second trimester maternal plasma sample
using the primer extension assay. Products from control reactions
containing primer only, unmethylated G allele or unmethylated A
allele are shown.
[0016] The sizes (nt) of the reaction products are shown at the
bottom. .cndot., unused primer; .quadrature. detected allele.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0017] In a first aspect, the present invention features methods
for differentiating DNA species originating from different
individuals in a biological sample. In preferred embodiments the
methods of the present invention are used to differentiate or
detect fetal DNA in a maternal sample or to differentiate DNA of an
organ donor from DNA of an organ recipient.
[0018] Those of skill in the art will appreciate that the
biological sample obtained from an individual may be taken from any
fluid or cell sample, however, in preferred embodiments the bodily
fluid is plasma or serum. In preferred embodiments, the DNA species
are differentiated by observing epigenetic differences in the DNA
species such as differences in DNA methylation. For instance, in
situations where one DNA species comes from a male, and one DNA
species comes from a female, the epigenetic marker may be the
inactivated X chromosome of the female individual. In such
embodiments, methylated DNA sequences on the inactivated X
chromosome may be used to detect DNA originating from the female
individual. In some embodiments, the epigenetic differences may be
analyzed inside cells. Further, in some embodiments, the epigenetic
differences may be analyzed using in-situ methylation-specific
polymerase chain reaction. Additionally, the epigenetic differences
may be used to sort or isolate cells from the respective
individuals or to purify DNA from the respective individuals. The
methods according to the present invention may be performed with or
without measuring the concentrations of DNA species, however, in
preferred embodiments, the concentrations of DNA species with the
respective epigenetic differences are measured. Such measuring of
concentrations involves measuring the respective DNA methylation
differences in embodiments wherein DNA methylation differences is
the epigenetic marker. In especially preferred embodiments, sodium
bisulfite is added to the biological sample or to the DNA species
directly to detect the DNA methylation differences. However, in
other embodiments a methylation-specific polymerase chain reaction,
as is well known to those skilled in the art, may be used to detect
the DNA methylation differences. In yet other embodiments, DNA
sequencing or primer extension may be used to detect the
methylation differences.
[0019] As used herein, the term "biological sample" is intended to
encompass any fluid or cellular sample or mixture thereof obtained
from a living organism. Specifically, the term includes tissue
biopsy, serum, plasma or amniotic fluid samples.
[0020] As used herein, the term "epigenetic difference" is intended
to encompass any molecular or structural difference other than the
primary nucleotide sequence. For instance, this may include
differences in methylation.
[0021] As used herein, the term "DNA" is intended to encompass any
sequence of more than one nucleotide such as polynucleotides, gene
fragments and complete gene sequences.
[0022] As used herein, the term "methylation-specific PCR" is used
to describe a method in which DNA is treated with sodium bisulfite
and then subjected to PCR amplification. This technique is based on
the principle that treating DNA with bisulfite results in
conversion of unmethylated cytosine residues into uracil.
Methylated cytosine residues, on the other hand, remain unchanged.
Thus, the DNA sequences of methylated and unmethylated genomic
regions following bisulfite conversion are different and
distinguishable by sequence-specific PCR primers.
[0023] The present invention utilizes the phenomenon of genomic
imprinting to overcome the limitations of the prior art. In genomic
imprinting, DNA sequences are modified biochemically, without
alteration in DNA sequence. If this process results in differential
modification of the fetal and maternal DNA, then this difference
can be exploited for the discrimination of fetal from maternal DNA
in maternal plasma and serum. This phenomenon can also be used for
the discrimination of fetal cells from maternal cells in the
cellular fraction of maternal blood. In addition, this principle
can also be used to detect maternal cells or DNA that has entered
into the body of the fetus/baby (Lo, et al., Blood 88 (11):4390-5
(1996); Lo, et al., Clin Chem, 46 (9):1301-9 (2000); Maloney et
al., J Clin Invest 104 (1):41-7 (1999). This phenomenon can also be
used in many other clinical scenarios wherein cells or DNA
sequences are found to be present inside the body of an individual,
such as following bone marrow transplantation (Lo et al., Br J
Haematol 89 (3):645-9 (1995)) or solid organ transplantation
(Starzl et al., Curr Opin Nephrol Hypertens 6 (3):292-8 (1997); Lo
et al., Lancet 351 (9112):1329-30 (1998); Zhang, Clin Chem 45
(10):1741-6 (1999)).
[0024] The present invention allows development of a
gender-independent and polymorphism-independent marker for fetal
DNA in maternal plasma/serum. To develop a gender-independent and
polymorphism-independent fetal marker, one can use DNA sequences
which are preferentially and specifically methylated in the
trophoblasts (Ohgane et al., Dev Genet, 22 (2):132-40 (1998)). This
overcomes the current limitation which can only easily detect the
presence of DNA from a male fetus in the plasma/serum of the mother
(by using the Y-chromosome as the target) (Lo, et al., Am J Hum
Genet, 62 (4):768 (1998). It provides detection methods separate
from relying on sequence differences in fetal and maternal DNA to
make such a distinction (Tang et al., Clin Chem 45 (11):2033-5
(1999); Pertl et al., Hum Genet 106:45-49 (2000)).
[0025] The development of molecular detection methods such as the
PCR has provided many powerful tools for the monitoring of
chimerism following bone marrow transplantation (BMT). One of the
most widely used PCR-based tests for the detection of post-BMT
chimerism in sex-mismatched cases is PCR for sequences on the Y
chromosome (Lo et al., Br J Haematol 89: 645-9 (1995). The
limitation of this strategy is that it can only be used in cases
wherein the donor is male and the recipient is female. The present
invention provides a system that can be applied to situations when
the donor is female and the recipient is male. The fact that the
phenomenon of Lyonization only exists in females, can be exploited
to develop a female-specific marker. In this phenomenon, one of the
two X chromosomes in a female individual is inactivated at random,
with methylation occurring in a number of the inactivated genes.
This therefore allows an assay for detecting female DNA in an
excess of male DNA and which can be applied to BMT with female
donors and male recipients.
[0026] In a second aspect, the present invention features methods
of detecting abnormalities in a fetus by detecting fetal DNA in a
biological sample obtained from a mother. The methods according to
the present invention provide for detecting fetal DNA in a maternal
sample by differentiating the fetal DNA from the maternal DNA based
upon epigenetic markers such as differences in DNA methylation.
Employing such methods, fetal DNA that is predictive of an anomaly
or a disease may be identified thereby providing methods for
prenatal diagnosis. These methods are applicable to any and all
pregnancy-associated conditions for which methylation changes
associated with a disease state is identified. Exemplary diseases
that may be diagnosed include, for example, preeclampsia, a
chromosomal aneuploidy, including but not limited to trisomy 21,
Prader-Willi Syndrome, and Angelman Syndrome.
[0027] As with the broader differentiating methods of the first
aspect of the invention, the biological sample obtained from the
mother is preferably plasma or serum. The differentiation between
maternal and fetal DNA may be performed with or without quantifying
the concentration of fetal DNA in maternal plasma or serum. In
embodiments, wherein the fetal DNA is quantified, the measured
concentration may be used to predict, monitor or diagnose a
pregnancy-associated disorder. In preferred embodiments, the
particular fetus-derived epigenetic mark is associated with a fetal
disorder, and in some embodiments an epigenetic characterisitic in
fetal cells in the placenta is used as a fetus-specific marker in
maternal plasma or serum.
[0028] The present invention utilizes differentially methylated
fetal DNA sequences, which do not need to be distinguishable in
terms of DNA sequence from maternal DNA, as markers for
non-invasive prenatal diagnosis. This novel approach can convert
fetus-mother pairs who are not informative in the conventional
approach, to being informative for prenatal diagnosis. Thus,
present invention provides a platform on which a new generation of
non-invasive prenatal tests can be built.
[0029] The methods of the present invention are based on the
detection of differently methylated DNA of fetal origin in the
plasma or serum of pregnant women. Differentially methylated DNA
sequences, which may contain single nucleotide polymorphism, are
preferably detected by methylation-specific polymerase chain
reaction (PCR); but in principle any detection method for
differentially methylated DNA can be used. This approach allows the
use of conventional uninformative fetal DNA markers for prenatal
diagnosis.
[0030] The present invention allows detecting or predicting the
presence of any disorders of the fetus or the mother which are
associated with a change in methylation status of a DNA sequence.
Examples include imprinting disorders such as Prader-Willi syndrome
(Kubota et al., Nat Genet 16 (1):16-7 (1997). The present invention
provides a new type of test for preeclampsia which has been
suggested to be an imprinting disorder (Graves, Reprod Fertil Dev
10 (1):23-9 (1998). The present invention further provides a new
type of test for chromosomal aneuploidies, including Down syndrome
(trisomy 21), which may be associated with methylation changes (Yu
et al., Proc Natl Acad Sci USA 94 (13):6862-7 (1997).
[0031] The present invention features using DNA methylation
differences between the mother and fetus thereby overcoming the
limitations of the prior art in the detection of fetal DNA in
maternal plasma.
[0032] In a third aspect, the present invention features methods
for differentiating DNA species originating from an organ donor
from those of an organ recipient. As with the broader
differentiating methods of the first aspect of the invention, the
biological sample obtained is preferably plasma or serum. The
differentiation between DNA from the organ donor and organ
recipient or potential organ donor and potential organ recipient
may be performed with or without quantifying the concentration of
DNA in the biological sample. This embodiment is particularly
useful in instances when the transplantation is a bone marrow
transplantation. Such measurements may be used to predict the
clinical progress of the transplantation recipient especially as
applied to organ rejection.
[0033] In a fourth aspect, the present invention features kits for
differentiating DNA species originating from different individuals
in a biological sample. Such kits are useful, for instance, for
differentiating or detecting the presence of fetal DNA in a
maternal biological sample or for differentiating DNA from an organ
donor or potential organ donor from that of an organ recipient or
potential organ recipient. The kits according to the present
invention comprise one or more reagents for ascertaining the
methylation status of the maternal DNA such as sodium bisulfite and
one or more reagents for detecting the presence of DNA such as a
gel. Additionally, such kits may include one or more reagents for
amplifying the amount of DNA present in the sample such as one or
more reagents for performing polymerase chain reaction
amplification. Such reagents are well known to those of skill in
the art. Further, such kits may include one or more apparatuses for
obtaining a maternal DNA sample. Such apparatuses are well known to
those skilled in the art. In particular the kits according to the
present invention may be used for diagnosing a disease caused all
or in part by a genetic anomaly such as a mutation, substitution or
deletion or duplication in all or part of a DNA sequence present in
a fetus. Exemplary diseases that may be diagnosed include, for
example, preeclampsia, a chromosomal aneuploidy, including but not
limited to trisomy 21, Prader-Willi Syndrome and Angelman
Syndrome.
EXAMPLE 1
Detection of Post-Bone Marrow Transplantation Chimerism Using a
Novel Epigenetic Approach
Materials and Methods
Subjects and Samples
[0034] Four male marrow transplantation recipients, who received
bone marrow from female donors, and 17 normal healthy subjects were
recruited in this study. Buffy coat (BC) from all recruited
EDTA-blood samples were harvested and stored at -20.degree. C. as
described (Lo et al., Am J Hum Genet 62:768-75 (1998).
DNA Isolation
[0035] DNA was extracted from the BC using a Nucleon DNA Extraction
Kit (Scotlabs) according to manufacturer's recommendations.
Bisulfite Conversion
[0036] Bisulfite modification of DNA samples was performed using a
CpGenome DNA Modification Kit (Intergen) as instructed by the
manufacturer. With bisulfite conversion, unmethylated cytosine
residues are converted to uracil while methylated cytosine residues
remain unchanged (Herman et al., Proc Natl Acad Sci USA 93:9821-6
(1996). The sequence difference between methylated and unmethylated
DNA following bisulfite conversion is then distinguished using
different PCR primers. 1 .mu.g of BC DNA was used in a bisulfite
conversion reaction.
Methylation-Specific PCR (MSP)
[0037] MSP assays were modified from the protocol as described by
Herman et al, supra. The primers M-for
(5'-GCGAGCGTAGTATTTTTCGGC-3') and M-rev
(5'-AACCAAATAACCTATAAAACCTCTACG-3') were designed for the
methylated sequence, while the primers U-for
(5'-GTTGTGAGTGTAGTATTTTTTGGT-3') and U-rev
(5'-CAAATAACCTATAAAACCTCTACA-3') were designed for the unmethylated
sequence. Five .mu.l bisulfite-treated DNA was added to a 50 .mu.l
PCR reaction containing 5 .mu.l 10.times. TaqMan buffer A (PE
Applied Biosystems), 2 mM MgCl.sub.2, 10 pmol dNTPs, 20 pmol each
of the corresponding MSP primers and 1.25 U AmpliTaq Gold DNA
polymerase (PE Applied Biosystems). Reaction mixtures were thermal
cycled (methylated allele: 95.degree. C. for 45 sec, 58.degree. C.
for 30 sec, 72.degree. C. for 20 sec; unmethylated allele:
95.degree. C. for 45 sec, 50.degree. C. for 30 sec, 72.degree. C.
for 20 sec) for 45 cycles, with an initial denaturing step of 8 min
at 95.degree. C. PCR products were then analyzed by agarose gel
electrophoresis.
Results
[0038] This experiment provides a MSP assay to detect methylated
and unmethylated DNA sequences of the androgen receptor gene. In
total, 6 male and 11 female health subjects were recruited. Of all
male control subjects, only the unmethylated androgen receptor gene
was detected in these samples as expected (FIG. 1A). By contrast,
both unmethylated and methylated androgen receptor gene DNA
sequences were observed in female control subjects (FIG. 1A). The
detection rates of methylated and unmethylated androgen receptor
genes in these female subjects were 100% and 82%, respectively.
When DNA samples were omitted from MSP assay, no positive signal
was observed (FIG. 1A). Interestingly, positive signals for both
methylated and unmethylated DNA sequences were observed in all male
sex-mismatched bone marrow transplantation recipients (100%),
indicating cells from female donor exist in the blood circulation
of male recipients.
[0039] These results demonstrate, for the first time that
methylated genes on the inactivated X chromosome from female
individuals can be used as a female-specific marker in chimerism
research. This assay is also applicable to the study of other types
of post-transplantation chimerisms involving mixture of male and
female cells or DNA. Examples include cellular chimerism following
solid organ transplantation (Starzl et al., Curr Opin Nephrol
Hypertens 6:292-8 (1997)), post-transplantation plasma DNA
chimerism (Lo et al., Lancet 351:1329-30 (1998)) and urinary DNA
chimerism (Zhang et al., Clin Chem 45:1741-6 (1995)). In addition,
there is also much recent interest in the passage of cells and DNA
from the mother into the fetus during pregnancy (Lo et al., Blood
88:4390-5. (1996); Maloney et al., J Clin Invest 104: 41-7 (1999);
Lo et al., Clin Chem 46:1301-9 (2000). The epigenetic markers
developed should also be of used in chimerism of maternal origin in
male offsprings.
[0040] The current assay may be developed into a quantitative
format, using for example, real-time PCR technology (Lo et al.,
Cancer Res 59:3899-903 (1999)). Such development would allow us to
monitor the levels of chimerism in a particular person. Clinically
such an assay might have a role in the monitoring of graft
acceptance in BMT. In the case of urinary or plasma DNA chimerism,
such an assay might also be used for the monitoring of graft
rejection.
EXAMPLE 2
Differential DNA Methylation Between Fetus and Mother as a Strategy
for Detecting Fetal DNA in Maternal Plasma
[0041] The present experiment demonstrates that by using a
differentially methylated region in the human IGF2-H19 locus as an
epigenetic marker in maternal plasma, detection of an allele that
the fetus has inherited from the mother is possible. These results
greatly expand the prenatal diagnostic possibilities of fetal DNA
in maternal plasma allowing development of a gender- and
polymorphism-independent fetal-specific marker in maternal plasma
and new strategies for the prenatal diagnosis of imprinting
disorders and certain chromosomal aneuploidies.
Materials and Methods
Subjects and Samples
[0042] Samples were collected from pregnant women with informed
consent. In total, 21 and 18 women in the second trimester (17-21
weeks) and third trimester (37-42 weeks) of pregnancy,
respectively, were recruited for this study. None of the recruited
subjects had preeclampsia or preterm labor in the current
pregnancy. EDTA maternal blood and fetal amniotic fluid samples
were collected from the second trimester cases as described
previously (Lo et al., Am J Hum Genet 62:768-775 (1998)). For the
third trimester cases, we collected EDTA maternal blood samples at
2 to 3 h before normal vaginal delivery. EDTA fetal cord blood
samples were also collected immediately after delivery as described
(Lo et al., Clin Chem 46:1903-1906 (2000)). Plasma and buffy coat
from all recruited blood samples were harvested and stored at
-20.degree. C. as described (Lo et al., Am J Hum Genet 62:768-775
(1998)), except that plasma samples were recentrifuged at 16,000 g.
Amniotic fluid samples were stored at 4.degree. C.
DNA Isolation
[0043] DNA was extracted from plasma and amniotic fluid samples
using a QIAamp Blood Kit (Qiagen). Typically, 800 .mu.l of plasma
or amniotic fluid was used for DNA extraction per column. An
elution volume of 50-110 .mu.L it was used. DNA was extracted from
the buffy coat using a Nucleon DNA Extraction Kit (Scotlabs)
according to manufacturer's recommendations.
Genotyping of the DMR Polymorphic Region
[0044] The DMR in the human IGF2-H19 locus contains two 450-bp
repeat and seven 400-bp repeat units (Nakagawa et al., Proc Natl
Acad Sci USA 98:591-596 (2001)) (FIG. 2). An A/G SNP within the DMR
(Nakagawa et al., supra) was selected as a marker in our
investigation (FIG. 2). Polymerase chain reaction (PCR) was used to
amplify the SNP in both maternal and fetal DNA samples. Primers
were designed using the sequence of the Homo sapiens H19 gene
(Genbank accession number AF125183). Typically, 2 to 5 .mu.l eluted
DNA, purified from maternal buffy coat, cord buffy coat or amniotic
fluid was added to a 25 .mu.l PCR reaction containing 2.5 .mu.l
10.times. TaqMan buffer A (PE Applied Biosystems), 3 mM MgCl.sub.2,
6.26 pmol dNTPs, 5 pmol primers (forward:
5'-ggACGGAATTGGTTGTAGTT-3'; reverse: 5'-AGGCAATTGTCAGTTCAGTAA-3')
and 0.625 U AmpliTaq Gold DNA polymerase (PE Applied Biosystems)
(95.degree. C. for 8 min followed by 35 cycles of 95.degree. C. for
1 min, 56.degree. C. for 20 sec, 72.degree. C. for 20 sec). For the
forward primer, the nucleotides in upper case corresponded to
positions 7927 to 7944 of the H19 sequence (Genbank accession
number AF125183). For the reverse primer, the nucleotides were
complementary to positions 8309 to 8329 of the H19 sequence. PCR
products were then analysed by agarose gel electrophoresis and DNA
sequencing.
Bisulfite Conversion
[0045] Bisulfite modification of DNA samples was performed using a
CpGenome DNA Modification Kit (Intergen) as instructed by the
manufacturer. With bisulfite conversion, unmethylated cytosine
residues would be converted to uracil; while methylated cytosine
residues would remain unchanged (Herman et al., Proc Natl Acad Sci
USA 93:9821-9826 (1996)). The sequence difference between
methylated and unmethylated DNA following bisulfite conversion
could then be distinguished using different PCR primers. In
general, 1 .mu.g of buffy coat DNA from maternal or cord blood, or
93 .mu.l eluted DNA purified from maternal plasma or amniotic fluid
was used in a bisulfite conversion reaction. Bisulfite-treated DNA
was then eluted in 25-50 .mu.l 1.mu. Tris-EDTA.
Methylation-Specific PCR (MSP)
[0046] MSP assays were modified from the protocol as described
(Herman et al. 1996). Five .mu.lbisulfite-treated DNA was added to
a 50 .mu.l PCR reaction containing 5 .mu.l 10X TaqMan buffer A (PE
Applied Biosystems), 2.5 mM MgCl.sub.2, 10 pmol dNTPs, 20 pmol each
of the corresponding MSP primers (FIGS. 2) and 1.25 U AmpliTaq Gold
DNA polymerase (PE Applied Biosystems). The primers M-for and M-rev
(FIG. 2) were designed for the methylated sequence, while the
primers U-for and U-rev (FIG. 2) were designed for the unmethylated
sequence. Reaction mixtures were thermal cycled (methylated allele:
95.degree. C. for 45 sec, 55.degree. C. for 20 sec, 72.degree. C.
for 20 sec; unmethylated allele: 95.degree. C. for 45 sec,
49.degree. C. for 20 sec, 72.degree. C. for 20 sec) for 50 (buffy
coat and amniotic fluid DNA) or 56 (plasma DNA) cycles, with an
initial denaturing step of 8 min at 95.degree. C. PCR products were
then analyzed by agarose gel electrophoresis. Reaction products
were purified using Microspin S-300 HR columns (Amersham Pharmacia)
for DNA sequencing or the primer extension assay.
DNA Sequencing
[0047] Purified PCR products were sequenced using an ABI prism
dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE
Applied Biosystems) and the corresponding forward primers of the
PCR products. Sequencing products were analysed using an ABI Prism
310 Genetic Analyser (PE Applied Biosystems).
Primer Extension Assay
[0048] Two .mu.l of the purified MSP product was added to a 25
.mu.l reaction containing 50 .mu.M ddATP (2',3'-dideoxyadenine
triphosphate), 50 .mu.M dGTP, 50 .mu.M dTTP, 0.2 pmol Cys-5-labeled
primer (5'-GGGTTATTTGGGAATAGGATATTTA-3'), 4 U Thermo Sequenase
(Amersham Pharmacia) and 1.43 .mu.l concentrated buffer. Reactions
were thermal cycled for 40 cycles (95.degree. C. for 30 sec,
51.degree. C. for 20 sec, 72.degree. C. for 20 sec). The
Cys-5-labeled primer was 25 nucleotides (nt) in length and the
polymorphic site was 2 nt away from the 3'-end of the primer. For
the A allele, the incorporation of the ddATP at this polymorphic
site would produce chain termination, thus resulting in an
extension product of 27 nt (i.e., 25+2 nt). For the G allele, chain
extension would continue until the next A residue which was 5 nt
away from the 3'-end of the primer, thus resulting in an extension
product of 30 nt (i.e., 25+5 nt). Reaction products were
electrophoresed using a 14% denaturing polyacrylamide gel and
analysed using an ALF Express Sequencer (Amersham Pharmacia). Data
were analysed by the AlleleLinks program (Amersham Pharmacia).
Results
Genotyping of DMR
[0049] Thirty-nine pregnant women were recruited in this study.
Maternal genotype at the SNP within the DMR (FIG. 2) was determined
by direct sequencing of PCR products from the buffy coat DNA. The
number of pregnant women with each of the possible genotypes were
17 (GG, 43.6%), 16 (AG, 41.0%) and 6 (AA, 15.4%).
Detection of Fetal DNA in Plasma from Women Heterozygous for a
Biallelic Polymorphism
[0050] The 16 women who were heterozygous (i.e., AG) for the SNP
were selected for further examination. As this is a biallelic
polymorphism, these women would not be considered informative at
this polymorphic locus for the detection of fetal DNA in maternal
plasma, based on previous criteria (Lo et al., Ann N Y Acad Sci
731:204-213 (1994); Bianchi Am J Hum Genet 62:763-764 (1998)). To
demonstrate that differential methylation at this genomic region
would allow us to overcome this limitation, maternal DNA was
bisulfite-treated and amplified by MSP using the primers shown in
FIG. 2. Similarly, fetal DNA isolated from amniotic fluid (2nd
trimester samples) or buffy coat of cord blood (3rd trimester
samples) was subjected to PCR and MSP to determine the imprinting
status of the fetal alleles.
[0051] Amongst the 16 selected cases, the methylated (i.e.,
paternally-inherited) alleles from four 3rd trimester and seven 2nd
trimester fetal samples were different from the methylated alleles
of the respective mothers (FIG. 3a,b; compare panels 1 and 2). To
test if this differential methylation between fetus and mother
would allow the fetal allele to be detected from maternal plasma,
maternal plasma DNA from these cases was subjected to bisulfite
conversion, followed by MSP. Interestingly, the
paternally-inherited methylated fetal allele could be detected in
two 3rd trimester and four 2nd trimester maternal plasma samples
(FIG. 3a,b; panels 3). To exclude the possibility that these
observations were simply due to the existence of aberrantly
methylated maternal DNA in maternal plasma, we collected a
postnatal maternal plasma sample (.about.3.5 years after delivery)
from one of the positive cases for further examination. We did not
observe the additional methylated allele in this postnatal sample
(FIG. 3a, panel 4), indicating that the additional methylated
allele in maternal sample during pregnancy was of fetal origin. In
addition, no positive signal was observed in the plasma of
non-informative cases (n=4, data not shown), thus further
demonstrating the specificity of this MSP assay. Taken together,
these data indicate that the use of differential methylation
between mother and fetus allows detecting fetal DNA in maternal
plasma, even in cases which are not considered informative with
existing criteria.
Detection of Fetal-Derived Maternally-Inherited DNA from Maternal
Plasma
[0052] We then tested if the use of differential methylation
between mother and fetus might allow us to detect an allele that
the fetus has inherited from the mother. This type of analysis has
previously been thought to be impossible (Lo et al., Ann N Y Acad
Sci 731:204-213 (1994); Bianchi, Am J Hum Genet 62:763-764 (1998)).
As the maternally-inherited allele was unmethylated, the primers
U-for and U-rev (FIG. 2) were used to amplify the unmethylated
allele following bisulfite conversion. Among the 16 analyzed cases,
three 3rd trimester and five 2nd trimester maternal samples were
informative. In these cases, the fetus possessed an unmethylated
allele that was different from the unmethylated allele of the
mother. These results implied that in these cases, the mother had
originally inherited the fetal allele from her father and then
passed on to the fetus. Of these 8 informative cases, only a weak
positive signal was observed in one of the 3rd trimester samples on
direct sequencing (FIG. 4a, compared panel 1 and panel 2).
[0053] We reasoned that the weak signal in this single positive
case and the low detection rate of the unmethylated fetal allele
from maternal plasma might be due to the low sensitivity of the
direct sequencing method. To enhance the sensitivity of detection,
we employed a more sensitive primer extension assay to detect the
unmethylated fetal allele from the MSP reaction products. As the
SNP was an A/G polymorphism, ddATP was used as a reaction substrate
in the primer extension assay. Extended reaction products from the
A and G alleles were 27 and 30 nt long, respectively. No fetal
specific reaction product was present in the corresponding maternal
buffy coat samples (FIG. 4b, c; maternal BC). Strikingly, fetal
specific extension products were observed in two 3rd trimester
(FIG. 4b, arrow) and one 2nd trimester (FIG. 4c, arrow) maternal
plasma samples, indicating the presence of unmethylated fetal DNA
in maternal plasma. As controls, none of the tested non-informative
cases was positive in this assay (n=5, data not shown). These
results demonstrated, for the first time, the feasibility of using
epigenetic markers to detect a fetal-derived maternally-inherited
DNA sequence from maternal plasma.
Discussion
[0054] These results demonstrate that the use of epigenetic markers
overcomes the conventional limitations of detecting fetal DNA in
maternal plasma. It is possible to detect a paternally-inherited
fetal allele, which is genetically indistinguishable from a
maternal allele, from the mother's plasma, by the use of epigenetic
differences between the mother and fetus. Likewise, it is possible
to detect a maternally-inherited fetal allele from maternal
plasma.
[0055] This novel epigenetic approach will therefore expand the
repertoire of disorders wherein fetal DNA in maternal plasma can be
used.
[0056] Even with the use of relatively insensitive methods such as
direct sequencing and primer extension, the present results
demonstrate that it is possible to detect differentially methylated
fetal DNA sequences from maternal plasma. There was a lower
sensitivity in the detection of the unmethylated fetal DNA in
maternal plasma (FIG. 4), as compared with the analogous assay for
the methylated allele (FIG. 3). Using more sensitive detection
systems, such as allele-specific PCR (Newton et al., Nucleic Acids
Res 17:2503-2516 (1989)) and real-time methylation-specific PCR (Lo
et al., Cancer Res 59:3899-3903 (1999); Eads et al., Nucleic Acids
Res 28:E32 (2000)), might enhance the sensitivity of plasma-based
epigenetic analysis. The development of real-time
methylation-specific PCR is particularly interesting as it opens up
the possibility of quantifying fetal-specific methylation in
maternal plasma, as has already been achieved for the detection of
tumor DNA in circulation (Kawakami et al., J Natl Cancer Inst
92:1805-1811 (2000)).
[0057] The possible introduction of fetal DNA in maternal plasma as
a routine prenatal diagnostic tool has raised questions with regard
to the need of a generic marker for circulating fetal DNA (Lo et
al., Am J Hum Genet 62:768-775 (1998); Avent et al, Vox Sang
78:155-162 (2000)). Most proposals for such a marker have thus far
focused on the use of genetic polymorphisms between the mother and
fetus (Tang et al., Clin Chem 45:2033-2035 (1999); Pertl et al.,
Hum Genet 106:45-49 (2000)). The present demonstration of the
feasibility of epigenetic markers for fetal DNA detection in
maternal plasma opens up a new approach for the development of a
gender-independent and polymorphism-independent fetal marker in
maternal plasma. One way wherein this can be achieved is to exploit
the phenomenon of tissue-specific methylation (Grunau et al., Hum
Mol Genet 9:2651-2663 (2000)). As the trophoblast has been
suggested to be the predominant cell population for releasing fetal
DNA into maternal plasma, the elucidation of trophoblast-specific
methylation patterns allows the development of a generic epigenetic
fetal marker in maternal plasma. Biologically, the use of
tissue-specific methylation markers may also allow one to directly
address the question as to what fetal cell types are responsible
for releasing fetal DNA into maternal plasma.
[0058] The epigenetic analysis of maternal plasma has obvious
applications to disorders associated with genomic imprinting, such
as the Prader-Willi syndrome (Pfeifer, Lancet 356:1819-1820
(2000)). This strategy may also have diagnostic potential for
disorders such as preeclampsia, wherein imprinted genes have been
hypothesized to play a role (Graves, Reprod Fertil Dev 10:23-29
(1998)).
[0059] The possible application of fetal DNA in maternal plasma for
the prenatal detection of fetal chromosomal aneuploidies is an
issue that has been keenly discussed since the discovery of the
phenomenon (Lo et al., Lancet 350:485-487 (1997); Bianchi, Am J Hum
Genet 62:763-764 (1998)). The finding of quantitative differences
between the circulating fetal DNA levels in aneuploid, compared
with euploid pregnancies (Lo et al., Clin Chem 45:1747-1751 (1999);
Zhong et al., Prenat Dia 20:795-798 (2000)) offers a method for
estimating the risk of fetal chromosomal aneuploidies from maternal
plasma. The recent discovery of apoptotic fetal cells in maternal
plasma ("plasma-derived cells") (Van Wijk et al., Clin Chem
46:729-731 (2000)) offers yet another approach for aneuploidy
detection from maternal plasma (Poon et al., Lancet 356:1819-1820
(2000)). Interestingly, the present data open up yet another
potential approach for the detection of fetal chromosomal
aneuploidies. This is based on the observation that aberrant DNA
methylation patterns may be associated with chromosomal aneuploidy
(Kuromitsu et al., Mol Cell Biol 17:707-712 (1997); Yu et al., Proc
Natl Acad Sci USA 94:6862-6867 (1997)). Hence it is possible to
develop epigenetic markers for detecting such aberrantly methylated
fetal DNA sequences from maternal plasma. Such markers provide
specificity compared with a simple quantitation of fetal DNA in
maternal plasma (Lo et al., Clin Chem 45:1747-1751 (1999); Zhong et
al., Prenat Diagn 20:795-798 (2000)) and better suitability to
large scale application compared with methods based on
"plasma-derived cells" (Poon et al., Lancet 356:1819-1820
(2000)).
[0060] Fetal epigenetic markers may also be used in the analysis of
fetal cells isolated from the cellular fraction of maternal blood.
This takes advantage of recent data showing that methylation
analysis could be performed in an in situ manner (Nuovo et al.,
Proc Natl Acad Sci USA 96:12754-12759 (1999)).
[0061] With the recent realization that fetomaternal trafficking is
a bidirectional process (Lo et al., Blood 88:4390-4395 (1996);
Maloney et al., J Clin Invest 104:41-47 (1999)), epigenetic markers
may also be used to investigate cellular and DNA transfer from the
mother to the fetus. Such an approach might also have applications
to the investigation of other types of chimerism, such as
post-transplantation hemopoietic chimerism (Starzl et al., Curr
Opin Nephrol Hypertens 6:292-298 (1997)) and urinary DNA chimerism
(Zhang et al., Clin Chem 45:1741-1746 (1999)).
[0062] With our increased understanding of the human genome and the
development of high throughput array-based technologies for
methylation analysis (Yan et al., Clin Cancer Res 6:1432-1438
(2000)), we expect that the number of usable fetal epigenetic
markers will rapidly increase over the next few years. Such a
development will provide a clinically relevant panel of fetal
epigenetic markers which can be used in a synergistic manner with
conventional genetic markers in maternal plasma.
Sequence CWU 1
1
11121DNAArtificial SequenceDescription of Artificial
Sequenceandrogen receptor gene methylated sequence
methylation-specific PCR (MSP) primer M-for 1gcgagcgtag tatttttcgg
c 21227DNAArtificial SequenceDescription of Artificial
Sequenceandrogen receptor gene methylated sequence
methylation-specific PCR (MSP) primer M-rev 2aaccaaataa cctataaaac
ctctacg 27324DNAArtificial SequenceDescription of Artificial
Sequenceandrogen receptor gene unmethylated sequence
methylation-specific PCR (MSP) primer U-for 3gttgtgagtg tagtattttt
tggt 24424DNAArtificial SequenceDescription of Artificial
Sequenceandrogen receptor gene unmethylated sequence
methylation-specific PCR (MSP) primer U-rev 4caaataacct ataaaacctc
taca 24520DNAArtificial SequenceDescription of Artificial
Sequencesingle nucleotide polymorphism (SNP) within differentially
methylated region (DMR) of human IGF2-H19 region amplification PCR
forward primer 5ggacggaatt ggttgtagtt 20621DNAArtificial
SequenceDescription of Artificial Sequencesingle nucleotide
polymorphism (SNP) within differentially methylated region (DMR) of
human IGF2-H19 region amplification PCR reverse primer 6aggcaattgt
cagttcagta a 21718DNAArtificial SequenceDescription of Artificial
Sequencesingle nucleotide polymorphism (SNP) site within
differentially methylated region of human IGF2-H19 region
methylated allele methylation-specific PCR (MSP) forward primer
M-for 7ttaattgggg ttcgttcg 18823DNAArtificial SequenceDescription
of Artificial Sequencesingle nucleotide polymorphism (SNP) site
within differentially methylated region of human IGF2-H19 region
methylated allele methylation-specific PCR (MSP) reverse primer
M-rev 8cccgacctaa aaatctaata cga 23922DNAArtificial
SequenceDescription of Artificial Sequencesingle nucleotide
polymorphism (SNP) site within differentially methylated region of
human IGF2-H19 region unmethylated allele methylation-specific PCR
(MSP) forward primer U-for 9ggtttgtttg tggaaatgtt tt
221023DNAArtificial SequenceDescription of Artificial
Sequencesingle nucleotide polymorphism (SNP) site within
differentially methylated region of human IGF2-H19 region
unmethylated allele methylation-specific PCR (MSP) reverse primer
U-rev 10cccaacctaa aaatctaata caa 231125DNAArtificial
SequenceDescription of Artificial Sequenceprimer extension assay
Cys-5-labeled primer 11gggttatttg ggaataggat attta 25
* * * * *