U.S. patent application number 16/168567 was filed with the patent office on 2019-02-07 for noninvasive prenatal genotyping of fetal sex chromosomes.
The applicant listed for this patent is The Chinese University of Hong Kong. Invention is credited to Kwan Chee Chan, Wai Kwun Rossa Chiu, Yuk Ming Dennis Lo, Bo Yin Tsui.
Application Number | 20190042693 16/168567 |
Document ID | / |
Family ID | 46457270 |
Filed Date | 2019-02-07 |
View All Diagrams
United States Patent
Application |
20190042693 |
Kind Code |
A1 |
Lo; Yuk Ming Dennis ; et
al. |
February 7, 2019 |
NONINVASIVE PRENATAL GENOTYPING OF FETAL SEX CHROMOSOMES
Abstract
Methods, apparatuses, and system are provided for analyzing a
maternal sample to determine whether a male fetus of a pregnant
female has inherited an X-linked mutation from the mother. A
percentage of fetal DNA in the sample is obtained, and cutoff
values for the two possibilities (fetus inherits mutant or normal
allele) are determined. A proportion of mutant alleles relative to
a normal allele on the X-chromosome can then be compared to the
cutoff values to make a classification of which allele is
inherited. Alternatively, a number of alleles from a target region
on the X-chromosome can be compared to a number of alleles from a
reference region on the X-chromosome to identify a deletion or
amplification. The fetal DNA percentage can be computed by counting
reactions with a fetal-specific allele, and correcting the number
to account for a statistical distribution among the reactions.
Inventors: |
Lo; Yuk Ming Dennis; (Hong
Kong, CN) ; Chiu; Wai Kwun Rossa; (Hong Kong, CN)
; Chan; Kwan Chee; (Hong Kong, CN) ; Tsui; Bo
Yin; (Hong Kong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Chinese University of Hong Kong |
Shatin |
|
HK |
|
|
Family ID: |
46457270 |
Appl. No.: |
16/168567 |
Filed: |
October 23, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13978358 |
Oct 3, 2013 |
10152568 |
|
|
PCT/IB2012/000015 |
Jan 5, 2012 |
|
|
|
16168567 |
|
|
|
|
61475632 |
Apr 14, 2011 |
|
|
|
61430032 |
Jan 5, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
G16B 40/00 20190201; C12Q 2600/156 20130101; G16B 20/50 20190201;
C12Q 2600/158 20130101; G16B 20/00 20190201; G16B 20/10
20190201 |
International
Class: |
G06F 19/18 20060101
G06F019/18; C12Q 1/6883 20060101 C12Q001/6883 |
Claims
1. A method for determining whether a male fetus of a pregnant
female has an X-linked mutation, wherein the pregnant female is
heterozygous for a mutant and a normal allele at a locus on the X
chromosome, the method comprising: receiving, by a computer system,
data from a plurality of reactions, each involving one or more
nucleic acid molecules from a biological sample, the biological
sample including cell-free nucleic acid molecules from the pregnant
female and from the male fetus, wherein the data includes: a first
set of quantitative data indicating a first amount of the mutant
allele at the locus; and a second set of quantitative data
indicating a second amount of the normal allele at the locus;
determining, by the computer system, a parameter from the first
amount and the second amount, wherein the parameter represents a
relative amount between the first and second amounts; obtaining, by
the computer system, a percentage Pf of fetal nucleic acid
molecules in the biological sample; calculating, by the computer
system, a first cutoff value for determining whether the fetus has
inherited the mutant allele at the locus, wherein the first cutoff
value is derived at least from a first proportion of k/(1+k-Pf),
where k is a number of mutant alleles on a mutant chromosome of the
pregnant female, k being an integer equal to or greater than one;
calculating, by the computer system, a second cutoff value for
determining whether the fetus has inherited the normal allele at
the locus, wherein the second cutoff value is derived at least from
a second proportion of [k(1-Pf)]/[1+k-kPf)]; and comparing, by the
computer system, the parameter to at least one of the first and
second cutoff values to determine a classification of whether the
fetus has inherited the mutant allele or the normal allele.
2. The method of claim 1, wherein the parameter is compared to the
first and second cutoff values.
3. The method of claim 2 wherein the classifications include
disease state, non-disease state, and non-classifiable.
4. The method of claim 1, wherein obtaining the percentage Pf
includes: correcting an experimentally derived percentage of fetal
nucleic acid molecules in the biological sample with an expected
statistical distribution of molecules in the plurality of
reactions.
5. The method of claim 1, wherein obtaining the percentage Pf
includes: detecting a first allele in the reactions, wherein the
first allele is shared by the pregnant female and fetus at a locus
where the pregnant female is homozygous and the fetus is either
heterozygous or hemizygous; calculating a Poisson-corrected
concentration Px with the equation [-ln((N-P1)/N)]*N, where N is
the total number of reactions analyzed, P1 is the number of
reactions positive for the first allele, and ln is the natural
logarithm; detecting a second allele in the reactions, wherein the
second allele is specific to the fetus; and calculating a
Poisson-corrected concentration Py with the equation
[-ln((N-P2)/N)]*N, where N is the total number of reactions
analyzed, and P2 is the number of reactions positive for the second
allele.
6. The method of claim 5, wherein the second allele is on
chromosome Y.
7. The method of claim 5, wherein the first allele is on chromosome
X.
8. The method of claim 5, wherein the fetal-specific allele is a
paternally-inherited allele on an autosome.
9. The method of claim 5, wherein the fetal-specific allele
includes a methylation marker specific to the fetus.
10. The method of claim 5, further comprising: calculating Pf as
[(2Py)/(Px+Py)]*100%.
11. The method of claim 1, wherein the first and second cutoff
values are determined using a sequential probability ratio test
(SPRT) to determine whether the fetus has inherited the mutant or
the normal nucleic acid sequence.
12. The method of claim 1, wherein an allele at a polymorphic site
linked to the mutant allele is located on the same maternal
haplotype as the mutant allele, and wherein the probability of
recombination between the polymorphic site and the mutant allele is
less than 1%.
13. The method of claim 1, wherein an allele at a polymorphic site
linked to the normal allele is located on the same maternal
haplotype as the normal allele, and wherein the probability of
recombination between the polymorphic site and the mutant allele is
less than 1%.
14. The method of claim 1, wherein the reactions include any one or
more of the following: sequencing reactions, optical analysis, and
hybridization using a fluorescent probe, or nanopore
sequencing.
15. The method of claim 1, wherein a reaction is an amplification
reaction.
16. The method of claim 15, wherein the reactions include
polymerase chain reactions.
17. The method of claim 15, wherein an average concentration is
less than one template molecule per reaction, and wherein a Poisson
distribution is used in determining the percentage Pf of fetal
nucleic acid molecules in the biological sample.
18. The method of claim 1, wherein the biological sample is plasma,
serum, or whole blood from a pregnant woman.
19. The method of claim 1, further comprising: displaying, by the
computer system, the classification of whether the fetus has
inherited the mutant allele or the normal allele based on comparing
the parameter to at least one of the first and second cutoff
values.
20. The method of claim 1, wherein the plurality of reactions is at
least 1,000 reactions.
21. The method of claim 1, further comprising: determining the
fetus has inherited the mutant allele.
22. The method of claim 1, wherein the X-linked mutation is a
mutation related to hemophilia, Duchenne muscular dystrophy,
X-linked adrenoleukodystrophy, Becker muscular dystrophy,
choroideremia, Hunter syndrome, Lesch Nyhan syndrome, Norrie's
syndrome, or ornithine transcarbamylase deficiency.
23. The method of claim 1, wherein the X-linked mutation is a
mutation related to hemophilia, and the method further comprising:
determining the fetus has inherited the mutant allele.
24. The method of claim 1, wherein the first amount is less than
1160, and the method further comprises determining the fetus has
inherited the mutant allele.
25. The method of claim 1, wherein: the parameter is a first
parameter; the method further comprising: determining based on the
first cutoff value and the second cutoff value that the fetus
cannot be classified as inheriting the mutant allele and cannot be
classified as inheriting the normal allele; receiving, by the
computer system, data from a second plurality of reactions, each
reaction involving one or more nucleic acid molecules from the
biological sample, wherein data from each of the one or more second
pluralities of reactions includes: a third set of quantitative data
indicating a third amount of the mutant allele at the locus; and a
fourth set of quantitative data indicating a fourth amount of the
normal allele at the locus; determining, by the computer system, a
second parameter from the first amount, the second amount, the
third amount, and the fourth amount, wherein the second parameter
represents a relative amount between the sum of the first amount
and the third amount and the sum of the second amount and the
fourth amount; comparing, by the computer system, the second
parameter to at least one of the first and second cutoff values to
classify the fetus as inheriting either the mutant allele or the
normal allele.
26. The method of claim 25, wherein the first plurality of
reactions and the second plurality of reactions total to less than
or equal to 13,770 reactions.
27. The method of claim 25, wherein: the first cutoff value is
based on a total number of reactions, and the second cutoff value
is based on the total number of reactions, the method further
comprising: updating the first cutoff value based on a total number
of the first plurality of reactions and the second plurality of
reactions, and updating the second cutoff value based on the total
number of the first plurality of reactions and the second plurality
of reactions.
28. The method of claim 26, wherein: updating the first cutoff
value comprises using sequential probability ratio test (SPRT), and
updating the second cutoff value comprises using SPRT.
29. The method of claim 1, wherein: the classification comprises a
probability of accuracy, the probability of accuracy determined by
how much the parameter exceeds at least one of the first and second
cutoff values.
30. The method of claim 1, further comprising: causing the
plurality of reactions to be performed.
31. A computer program product comprising a non-transitory computer
readable medium storing a plurality of instructions for controlling
an apparatus to perform the method of claim 1.
32. A method for determining whether a male fetus of a pregnant
female has an X-linked mutation, the method comprising: receiving,
by a computer system, data from a plurality of reactions, each
involving one or more nucleic acid molecules from a biological
sample, the biological sample including cell-free nucleic acid
molecules from the pregnant female and from the male fetus, wherein
the pregnant female is homozygous for an allele at a locus on the X
chromosome, has a mutation of an amplification of the allele on a
mutant X chromosome, the mutant X chromosome having a normal copy
of the allele at the locus and one or more additional copies of the
allele, and has a normal X chromosome having a normal copy of the
allele at the locus, wherein the data includes: a first set of
quantitative data indicating a first amount of an additional
junction created by the one or more additional copies of the
allele; and a second set of quantitative data indicating a second
amount of a normal junction created by the normal copy of the
allele on both X chromosomes; determining, by the computer system,
a parameter from the first amount and the second amount, wherein
the parameter represents a relative amount between the first and
second amounts; obtaining, by the computer system, a percentage Pf
of fetal nucleic acid molecules in the biological sample;
calculating, by the computer system, a first cutoff value for
determining whether the fetus has inherited the mutant X
chromosome, wherein the first cutoff value is derived at least from
a first proportion of n/(n+1-Pf), where n is the number of
additional copies of the allele, n being an integer equal to or
greater than one; calculating, by the computer system, a second
cutoff value for determining whether the fetus has inherited the
normal X chromosome, wherein the second cutoff value is derived at
least from a second proportion of [n(1-Pf)/[n+2-Pf(n+1)]; and
comparing, by the computer system, the parameter to at least one of
the first and second cutoff values to determine a classification of
whether the fetus has inherited the mutant X chromosome or the
normal X chromosome.
33. The method of claim 32, further comprising: determining the
fetus has inherited the mutant X chromosome.
34. The method of claim 33, wherein the mutant X chromosome is
related to hemophilia.
35. The method of claim 32, further comprising: causing the
plurality of reactions to be performed.
36. A method for determining whether a male fetus of a pregnant
female has an X-linked mutation, wherein the pregnant female is
heterozygous for a mutation and a normal allele at a target region
on the X chromosome, wherein the mutation is a deletion or an
amplification of the target region, the method comprising:
receiving, by a computer system, data from a plurality of
reactions, each involving one or more nucleic acid molecules from a
biological sample, the biological sample including cell-free
nucleic acid molecules from the pregnant female and from the male
fetus, wherein the data includes: a first set of quantitative data
indicating a first amount of the nucleic acid molecules that are
from the target region; and a second set of quantitative data
indicating a second amount of the nucleic acid molecules that are
from a reference region on the X chromosome; determining, by the
computer system, a parameter from the first amount and the second
amount, wherein the parameter represents a relative amount between
the first and second amounts; obtaining, by the computer system, a
percentage Pf of fetal nucleic acid molecules in the biological
sample; calculating, by the computer system, a first cutoff value
for determining whether the fetus has inherited the mutation, the
first cutoff value being dependent on the percentage Pf;
calculating, by the computer system, a second cutoff value for
determining whether the fetus has inherited the normal allele, the
second cutoff value being dependent on the percentage Pf; and
comparing, by the computer system, the parameter to at least one of
the first and second cutoff values to determine a classification of
whether the fetus has inherited the mutation or the normal
allele.
37. The method of claim 36, wherein the mutation is an
amplification, wherein the first cutoff value is determined based
on the assumption that a ratio of the first amount to the second
amount is increased when compared with a corresponding ratio of a
non-pregnant woman carrying the same amplification mutation, and
the second cutoff value is based on the assumption that the ratio
of the first amount to second amount is decreased when compared
with the corresponding ratio of a non-pregnant woman carrying the
same amplification mutation.
38. The method of claim 36, wherein the mutation is a deletion,
wherein the second cutoff value is determined based on the
assumption that a ratio of the first amount to the second amount is
increased when compared with a corresponding ratio of a
non-pregnant woman carrying the same deletion mutation, and the
first cutoff value is based on the assumption that the ratio of the
first amount to the second amount is decreased when compared with
the corresponding ratio of a non-pregnant woman carrying the same
deletion mutation.
39. The method of claim 36, wherein the mutation is a deletion,
wherein the second cutoff value is derived at least from a first
proportion of 1/(2-Pf), and wherein the first cutoff value is
derived at least from a second proportion of (1-Pf)/(2-Pf).
40. The method of claim 36, wherein the mutation is a duplication,
wherein the second cutoff value is derived at least from a first
proportion of (3-Pf)/(2-Pf), and wherein the first cutoff value is
derived at least from a second proportion of (3-2Pf)/(2-Pf).
41. The method of claim 36, wherein obtaining the percentage Pf
includes: correcting an experimentally derived percentage of fetal
nucleic acid molecules in the biological sample with an expected
statistical distribution of molecules in the plurality of
reactions.
42. The method of claim 36, further comprising: determining the
fetus has inherited the mutation.
43. The method of claim 42, wherein the mutation is related to
hemophilia.
44. The method of claim 36, further comprising: causing the
plurality of reactions to be performed.
45. A method of obtaining a percentage Pf of fetal nucleic acid
molecules in a biological sample from a female pregnant with a
fetus, the method comprising: receiving, by a computer system, data
from a plurality of reactions, each involving a plurality of
nucleic acid molecules from the biological sample, the biological
sample including cell-free nucleic acid molecules from the pregnant
female and from the fetus; detecting, by the computer system, a
first allele in the reactions, wherein the first allele is shared
by the pregnant female and fetus at a locus where the pregnant
female is homozygous and the fetus is either heterozygous or
hemizygous, wherein detecting the first allele comprises aligning
sequence tags to a reference genome to identify tags aligning to
the first allele; calculating, by the computer system, a corrected
concentration Px of the first allele based on a number of reactions
positive for the first allele, where Px is corrected for an
expected statistical distribution of the first allele in the
plurality of reactions; detecting, by the computer system, a second
allele in the reactions, wherein the second allele is specific to
the fetus, wherein detecting the second allele comprises aligning
sequence tags to a reference genome to identify tags aligning to
the second allele; calculating, by the computer system, a corrected
concentration Py of the second allele based on a number of
reactions positive for the second allele, where Py is corrected for
an expected statistical distribution of the second allele in the
plurality of reactions; and calculating, by the computer system, Pf
using [(2Py)/(Px+Py)].
46. The method of claim 45, wherein Pf equals
[(2Py)/(Px+Py)]*100%.
47. The method of claim 45, wherein the statistical distribution is
Poisson, and wherein the Poisson-corrected concentration Px uses
the equation [-ln((N-P1)/N)]*N, where N is the total number of
reactions analyzed, P1 is the number of reactions positive for the
first allele, and ln is the natural logarithm, and wherein the
Poisson-corrected concentration Py uses the equation
[-ln((N-P2)/N)]*N, where N is the total number of reactions
analyzed, and P2 is the number of reactions positive for the second
allele.
48. The method of claim 45, wherein the data includes: a first set
of quantitative data indicating a first amount of an allele at a
polymorphic site linked to the first allele; and a second set of
quantitative data indicating a second amount of an allele at a
polymorphic site linked to the second allele, the method further
comprising: determining a parameter from the two data sets;
determining a first cutoff value for determining whether the fetus
has inherited a mutant nucleic acid sequence, wherein the first
cutoff value is determined based on the percentage Pf; determining
a second cutoff value for determining whether the fetus has
inherited the normal nucleic acid sequence, wherein the second
cutoff value is determined based on the percentage Pf; comparing
the parameter to at least one of the first and second cutoff
values; and based on the comparison, determining a classification
of whether the fetus has inherited the mutant or the normal nucleic
acid sequence.
49. The method of claim 45, further comprising: causing the
plurality of reactions to be performed.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 13/978,358, entitled "Noninvasive
Prenatal Genotyping Of Fetal Sex Chromosomes" by Lo et al., with a
371(c) date of Oct. 3, 2013, which is a U.S. National Phase of
International Patent Application No. PCT/IB2012/000015, entitled
"Noninvasive Prenatal Genotyping Of Fetal Sex Chromosomes" by Lo et
al. (008310PC), filed Jan. 5, 2012, which claims priority from and
is a non-provisional application of U.S. Provisional Application
No. 61/430,032, entitled "Noninvasive Prenatal Genotyping Of Fetal
Sex Chromosomes" by Lo et al. (008300US), filed Jan. 5, 2011; and
U.S. Provisional Application No. 61/475,632, entitled "Noninvasive
Prenatal Genotyping Of Fetal Sex Chromosomes" by Lo et al.
(008301US), filed Apr. 14, 2011, the entire contents of which are
herein incorporated by reference for all purposes.
[0002] This application is related to commonly owned U.S. patent
application Ser. No. 12/178,116 entitled "Determining a Nucleic
Acid Sequence Imbalance" by Lo et al. (005210US), filed Jul. 23,
2008, the disclosure of which is incorporated by reference in its
entirety.
REFERENCE TO A "SEQUENCE LISTING" SUBMITTED AS ASCII TEXT FILES VIA
EFS-WEB
[0003] The Sequence Listing written in file
080015-008320US-1100023_SequenceListing.txt created on Oct. 22,
2018, 12,731 bytes, machine format IBM-PC, MS-Windows operating
system, in accordance with 37 C.F.R. .sctn..sctn. 1.821- to 1.825,
is hereby incorporated by reference in its entirety for all
purposes.
BACKGROUND
[0004] Hemophilias A and B are caused by heterogeneous mutations in
the genes on chromosome X that encode for the coagulation factor
VIII (F8) (Kemball-Cook G, Tuddenham E G, Nucleic Acids Res.,
25:128-132 (1997)) and coagulation factor IX (F9) (Giannelli F,
Green P M, Sommer S S, et al., Nucleic Acids Res., 26:265-268
(1998)), respectively. There is a 25% chance for a pregnant
hemophilia carrier to have an affected male fetus in each
pregnancy. Prenatal diagnosis is an important aspect of
reproductive choices for women in families with hemophilia (Lee C
A, Chi C, Pavord S R, et al., Haemophilia., 12:301-336 (2006)). In
addition, it is also beneficial for appropriate obstetric
management during labor and delivery as prolonged labor, invasive
monitoring techniques and instrumental deliveries should be avoided
in affected fetuses to minimize potential fetal and neonatal
hemorrhagic complications (Lee C A, Chi C, Pavord S R, et al.,
Haemophilia., 12:301-336 (2006)). Therefore, the development of a
noninvasive prenatal diagnostic approach for hemophilia is
beneficial to both obstetricians and hemophilia families.
[0005] Current prenatal diagnostic methods for sex-linked diseases
are typically invasive and pose a risk to the fetus. The discovery
of cell-free fetal DNA in maternal plasma has offered new
opportunities for noninvasive prenatal diagnosis (Lo Y M D et al.,
Lancet., 350:485-487 (1997); Lo Y M D, Chiu R W K, Nat Rev Genet.,
8:71-77 (2007)). A number of promising clinical applications have
been developed based on the detection of paternally inherited
genetic traits in maternal plasma. For example, the noninvasive
detection of fetal sex and RHD status are useful for the clinical
management of sex-linked diseases and RhD incompatibility
(Bustamante-Aragones A et al., Haemophilia., 14:593-598 (2008);
Finning K et al., BMJ., 336:816-818 (2008)). For monogenic diseases
such as achondroplasia and .beta.-thalassemia, the detection of the
presence or absence of paternally inherited mutations in maternal
plasma would allow one to diagnose autosomal dominant diseases or
exclude autosomal recessive diseases of the fetuses, respectively
(Saito H et al., Lancet., 356:1170 (2000); Chiu R W K et al.,
Lancet., 360:998-1000 (2002); Ding C et al., Proc Nail Acad Sci
USA., 101:10762-10767 (2004)).
[0006] Despite the rapid development of the field, it has remained
difficult to detect fetal alleles that are inherited from mothers
who are carriers for the mutations. The difficulty is caused by the
coexistence of fetal and maternal DNA in maternal plasma, and the
maternally inherited fetal allele is indistinguishable from the
background maternal DNA (Lo Y M D, Chiu R W K, Nat Rev Genet.,
8:71-77 (2007)).
[0007] Therefore, it is desirable to provide accurate and efficient
methods for determining whether a male fetus has inherited an
X-linked mutation.
BRIEF SUMMARY
[0008] Methods, apparatuses, and system are provided for analyzing
a maternal sample to determine whether a male fetus of a pregnant
female has inherited an X-linked mutation from the mother. A
percentage of fetal DNA in the sample is obtained, and cutoff
values for the two possibilities (fetus inherits mutant or normal
allele) are determined. A proportion of mutant alleles relative to
a normal allele on the X-chromosome can then be compared to the
cutoff values to make a classification of which allele is
inherited. Alternatively, a number of alleles from a target region
on the X-chromosome can be compared to a number of alleles from a
reference region on the X-chromosome to identify a deletion or
amplification. The fetal DNA percentage can be computed by counting
reactions with a fetal-specific allele, and correcting the number
to account for a statistical distribution among the reactions.
[0009] According to one embodiment, a method is provided for
determining whether a male fetus of a pregnant female has an
X-linked mutation. The pregnant female is heterozygous for a mutant
and a normal allele at a locus on the X chromosome. Data is
received from a plurality of reactions, each involving one or more
nucleic acid molecules from a biological sample. The biological
sample includes nucleic acid molecules from the pregnant female and
from the male fetus. The data includes a first set of quantitative
data indicating a first amount of the mutant allele at the locus
and a second set of quantitative data indicating a second amount of
the normal allele at the locus. A parameter is determined from the
first amount and the second amount, where the parameter represents
a relative amount between the first and second amounts. A
percentage Pf of fetal nucleic acid molecules in the biological
sample is obtained. A first cutoff value for determining whether
the fetus has inherited the mutant allele at the locus is
calculated, where the first cutoff value is derived at least from a
first proportion of k/(1+k-Pf), where k is a number of mutant
alleles on a mutant chromosome of the pregnant female, k being an
integer equal to or greater than one. A second cutoff value for
determining whether the fetus has inherited the normal allele at
the locus is calculated, where the second cutoff value is derived
at least from a second proportion of [k(1-Pf)]/[1+k-kPf)]. The
parameter is compared to at least one of the first and second
cutoff values to determine a classification of whether the fetus
has inherited the mutant allele or the normal allele.
[0010] According to another embodiment, a method is provided for
determining whether a male fetus of a pregnant female has an
X-linked mutation. The pregnant female is heterozygous for a
mutation and a normal allele at a target region on the X
chromosome. The mutation is a deletion or an amplification of the
target region. Data from a plurality of reactions is received. Each
reaction involves one or more nucleic acid molecules from a
biological sample. The biological sample includes nucleic acid
molecules from the pregnant female and from the male fetus. The
data includes a first set of quantitative data indicating a first
amount of the nucleic acid molecules that are from the target
region and a second set of quantitative data indicating a second
amount of the nucleic acid molecules that are from a reference
region on the X chromosome. A parameter is determined from the
first amount and the second amount, where the parameter represents
a relative amount between the first and second amounts. A
percentage Pf of fetal nucleic acid molecules in the biological
sample is obtained. A first cutoff value for determining whether
the fetus has inherited the mutation is calculated. The first
cutoff value is dependent on the percentage Pf. A second cutoff
value for determining whether the fetus has inherited the normal
allele is calculated. The second cutoff value is dependent on the
percentage Pf. The parameter is compared to at least one of the
first and second cutoff values to determine a classification of
whether the fetus has inherited the mutation or the normal
allele.
[0011] According to another embodiment, a method of obtaining a
percentage Pf of fetal nucleic acid molecules in a biological
sample from a female pregnant with a fetus. Data is receivied from
a plurality of reactions. Each reaction involves a plurality of
nucleic acid molecules from a biological sample, which includes
nucleic acid molecules from the pregnant female and from the fetus.
A first allele is detected in the reactions. The first allele is
shared by the mother and fetus at a locus where the pregnant female
is homozygous and the fetus is either heterozygous or hemizygous. A
corrected concentration Px of the first allele is calculated based
on a number of reactions positive for the first allele, where Px is
corrected for an expected statistical distribution of the first
allele in the plurality of reactions. A second allele is detected
in the reactions, where the second allele is specific to the fetus.
A corrected concentration Py of the second allele is calculated
based on a number of reactions positive for the second allele. Py
is corrected for an expected statistical distribution of the second
allele in the plurality of reactions. The fetal percentage Pf is
then calculated using [(2Py)/(Px+Py)].
[0012] Other embodiments are directed to systems, and computer
readable media associated with methods described herein.
[0013] A better understanding of the nature and advantages of the
present invention may be gained with reference to the following
detailed description and the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a flowchart illustrating a method 100 for
analyzing a maternal biological sample to diagnose an X-linked
disorder in a fetus according to embodiments of the present
invention.
[0015] FIG. 2A illustrates the two possibilities of the fetus
inheriting the mutant allele or the normal allele. FIG. 2B shows a
plot 250 of cutoff values for classifying a sample as obtained
using sequential probability ratio test (SPRT) according to
embodiments of the present invention
[0016] FIG. 3 is a flowchart illustrating a method 300 for
determining whether a male fetus of a pregnant female has an
X-linked mutation according to embodiments of the present
invention.
[0017] FIG. 4 illustrates a method 400 for determining whether a
male fetus has inherited an X-linked mutation according to
embodiments of the present invention.
[0018] FIG. 5A shows a table 500 illustrating a dosage imbalance
between mutant and wild-type alleles for mutations on chromosome X.
FIG. 5B illustrates a first scenario for detecting an amplification
when the pregnant subject is heterozygous at the locus of interest.
FIG. 5C illustrates a second scenario for detecting an
amplification when the pregnant subject is homozygous at the locus
of interest.
[0019] FIG. 6 is a flowchart illustrating a method 600 for
determining whether a male fetus of a pregnant female has an
X-linked mutation.
[0020] FIG. 7 is a table 700 showing a dosage imbalance between the
target and the reference loci for deletion and duplication
mutations on chromosome X.
[0021] FIG. 8 is a flowchart illustrating a method 800 for
obtaining a percentage Pf of fetal nucleic acid molecules in a
biological sample from a female pregnant with a fetus according to
embodiments of the present invention.
[0022] FIG. 9 shows a table 900 with clinical information of the
seven pregnant women who are carriers of hemophilia mutations.
[0023] FIG. 10 is a table 1000 showing oligonucleotide sequences
and real-time PCR conditions for the allele-discriminative assays
(SEQ ID NO: 1-36).
[0024] FIG. 11 is a table 1100 showing fetal genotyping for
rs6528633 in maternal plasma by digital RMD.
[0025] FIG. 12 shows the validation of digital RMD assays with
artificial DNA mixtures.
[0026] FIG. 13 is a table 1300 showing non-invasive detection of
fetal hemophilia mutations in maternal plasma by digital RMD.
[0027] FIG. 14 shows plots of SPRT analysis for fetal hemophilia
mutations in maternal plasma samples. Case numbers are indicated at
the top of the graphs. P.sub.r, proportion of positive wells
containing the mutant allele.
[0028] FIG. 15 shows digital RMD result for maternal plasma samples
from normal pregnancies.
[0029] FIG. 16 shows a block diagram of an example computer system
1600 usable with system and methods according to embodiments of the
present invention.
DEFINITIONS
[0030] The term "biological sample" as used herein refers to any
sample that is taken from a subject (e.g., a human, such as a
pregnant woman) and contains one or more nucleic acid molecule(s)
of interest.
[0031] The term "nucleic acid" or "polynucleotide" refers to a
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and a polymer
thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogs of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
The term nucleic acid is used interchangeably with gene, cDNA,
mRNA, small noncoding RNA, micro RNA (miRNA), Piwi-interacting RNA,
and short hairpin RNA (shRNA) encoded by a gene or locus.
[0032] The term "gene" means the segment of DNA involved in
producing a polypeptide chain. It may include regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0033] The term "reaction" as used herein refers to any process
involving a chemical, enzymatic, or physical action that is
indicative of the presence or absence of a particular
polynucleotide sequence of interest. An example of a "reaction" is
an amplification reaction such as a polymerase chain reaction
(PCR). Another example of a "reaction" is a sequencing reaction,
either by synthesis, ligation, hybridization or degradation. An
"informative reaction" is one that indicates the presence of one or
more particular polynucleotide sequence of interest, and in one
case where only one sequence of interest is present. The term
"well" as used herein refers to a reaction at a predetermined
location within a confined structure, e.g., a well-shaped vial,
cell, chamber in a PCR array, a droplet in an emulsion, a particle,
a nanopore or an area on a surface.
[0034] The term "overrepresented nucleic acid sequence" as used
herein refers to the nucleic acid sequence among two sequences of
interest (e.g., a clinically relevant sequence and a background
sequence) that is in more abundance than the other sequence in a
biological sample.
[0035] The term "based on" as used herein means "based at least in
part on" and refers to one value (or result) being used in the
determination of another value, such as occurs in the relationship
of an input of a method and the output of that method. The term
"derive" as used herein also refers to the relationship of an input
of a method and the output of that method, such as occurs when the
derivation is the calculation of a formula.
[0036] The term "quantitative data" as used herein means data that
are obtained from one or more reactions and that provide one or
more numerical values. For example, the number of wells that show a
fluorescent marker for a particular sequence would be quantitative
data.
[0037] The term "parameter" as used herein means a numerical value
that characterizes a quantitative data set and/or a numerical
relationship between quantitative data sets. For example, a ratio
(or function of a ratio) between a first amount of a first nucleic
acid sequence and a second amount of a second nucleic acid sequence
is a parameter.
[0038] As used herein, the term "locus" or its plural form "loci"
is a location or address of any length of nucleotides (or base
pairs) which has a variation across genomes. The term "alleles"
refers to alternative DNA sequences at the same physical genomic
locus, which may or may not result in different phenotypic traits.
In any particular diploid organism, with two copies of each
chromosome (except the sex chromosomes in a male human subject),
the genotype for each gene comprises the pair of alleles present at
that locus, which are the same in homozygotes and different in
heterozygotes. A population or species of organisms typically
includes multiple alleles at each locus among various individuals.
A genomic locus where more than one allele is found in the
population is termed a polymorphic site. Allelic variation at a
locus is measurable as the number of alleles (i.e., the degree of
polymorphism) present, or the proportion of heterozygotes (i.e.,
the heterozygosity rate) in the population. As used herein, the
term "polymorphism" refers to any inter-individual variation in the
human genome, regardless of its frequency. Examples of such
variations include, but are not limited to, single nucleotide
polymorphisms, simple tandem repeat polymorphisms,
insertion-deletion polymorphisms, mutations (which may be disease
causing) and copy number variations.
[0039] The term "cutoff value" as used herein means a numerical
value whose value is used to arbitrate between two or more states
(e.g. diseased and non-diseased) of classification for a biological
sample. For example, if a parameter is greater than the cutoff
value, a first classification of the quantitative data is made
(e.g. diseased state); or if the parameter is less than the cutoff
value, a different classification of the quantitative data is made
(e.g. non-diseased state).
[0040] The term "imbalance" as used herein means any significant
deviation as defined by at least one cutoff value in a quantity of
the clinically relevant nucleic acid sequence from a reference
quantity. For example, the reference quantity could be a ratio of
3/5, and thus an imbalance would occur if the measured ratio is
1:1.
[0041] The term "sequenced tag" as used herein refers to a string
of nucleotides sequenced from any part or all of a nucleic acid
molecule. For example, a sequenced tag may be a short string of
nucleotides sequenced from a nucleic acid fragment, a short string
of nucleotides at both ends of a nucleic acid fragment, or the
sequencing of the entire nucleic acid fragment that exists in the
biological sample. A nucleic acid fragment is any part of a larger
nucleic acid molecule. A fragment (e.g. a gene) may exist
separately (i.e. not connected) to the other parts of the larger
nucleic acid molecule.
DETAILED DESCRIPTION
[0042] Current prenatal diagnostic methods for sex-linked diseases
are typically invasive and pose a risk to the fetus. Cell-free
fetal DNA analysis in maternal plasma provides a noninvasive means
of assessing fetal sex in such pregnancies. However, the disease
status of male fetuses remains unknown if mutation-specific
confirmatory analysis is not performed. Here we have developed a
noninvasive tests to diagnose if the fetus has inherited a
causative mutation for sex-linked disease from its mother. One
strategy is based on a relative mutation dosage (RMD) approach
which we have previously established for determining the mutational
status of fetuses for autosomal disease mutations. The RMD method
is used to deduce if a fetus has inherited a sex-linked mutation on
chromosome X by detecting if the concentration of the mutant or
wild-type allele is overrepresented in the plasma of heterozygous
women carrying male fetuses.
[0043] Embodiments provide the application of the RMD approach in
prenatal diagnosis of X-linked disorders, e.g., hemophilia. A
difference between the RMD analyses for autosomal diseases and
X-linked diseases is that for the former there are three possible
fetal genotypes (i.e. homozygous normal, homozygous mutant, and
heterozygous) while for the latter there are only two possible
fetal genotypes. In the context of X-linked diseases, a male fetus
possesses only one chromosome X and thus it would be of either
mutant or wild-type genotype. The two outcomes for X-linked
diseases, as compared with the three outcomes for autosomal
diseases, can make the RMD approach more robust for X-linked
diseases for a given degree of analytical precision. Embodiments
can also be used for other sex-linked diseases, including but not
limited to Duchenne muscular dystrophy, X-linked
adrenoleukodystrophy, Becker muscular dystrophy, choroideremia,
Hunter syndrome, Lesch Nyhan syndrome, Norrie's syndrome and
ornithine transcarbamylase deficiency.
[0044] We illustrate the concept using hemophilia, a X-linked
bleeding disorder, as an example. We correctly detected fetal
genotypes for hemophilia mutations in all of the 12 studied
maternal plasma samples obtained from pregnancies at-risk of
hemophilia (a sex-linked disease) from as early as the 11.sup.th
week of gestation. This development would make the decision to
undertake prenatal testing less traumatic and safer for at-risk
families.
I. DETERMINING SEX-LINKED MUTATION
[0045] FIG. 1 is a flowchart illustrating a method 100 for
analyzing a maternal biological sample to diagnose an X-linked
disorder in a fetus according to embodiments of the present
invention. Method 100 is noninvasive and can use DNA circulating in
the maternal biological sample.
[0046] In step 110, a pregnant subject with a known mutation on an
X chromosome is identified. The mutation may be of any type as
described herein, such as hemophilia. The mutation may be
determined in a variety of ways, such as DNA sequencing, Southern
blot analysis, PCR (including allele-specific PCR), melting curve
analysis, etc. The mutation is such that only one of the X
chromosomes of the pregnant subject has the mutation, i.e., the
pregnant subject is heterozygous at a locus associated with the
mutation. Embodiments can also be applied for the noninvasive
prenatal diagnosis of other sex-linked disorders involving point
mutations or sequence deletion, duplication or inversion, for
examples, choroideremia and Norrie's syndrome.
[0047] In step 120, a biological sample of the pregnant subject is
received. The sample may be any biological sample that contains
fetal nucleic acids, such as plasma, urine, serum, and saliva. For
example, maternal plasma sample can be collected from a pregnant
carrier receiving obstetric care.
[0048] In step 130, the sex of the fetus is determined. The sex can
be determined by detecting X and Y chromosomes. Through the
detection of chromosome Y DNA sequences in maternal plasma, male
fetuses could be identified with an accuracy of greater than 97%
from the 7.sup.th week of gestation onwards. Unnecessary invasive
testing could be avoided for female fetuses, as they are either
unaffected or are disease carriers.
[0049] In step 140, the fetus is determined to be female, and then
no further analysis is performed at step 145. Female fetuses are
affected as carriers, except rare scenarios like skewed
X-inactivation.
[0050] In step 150, the fetus is determined to be male, and then in
step 155, DNA fragments on the X chromosome are analyzed. In one
embodiment, a fetal mutation detection is performed by a relative
mutation dosage (RMD) technique, which is described in more detail
below. In another embodiment, a fetal mutation of a deletion or
amplification is detected by comparing an amount of alleles at a
target region (which includes the mutation in the mother) to an
amount of alleles at a reference region, which is normal in the
mother.
[0051] In step 157, a determination that the fetus did not inherit
the mutated X chromosome of the maternal subject can be made. In
step 159, a determination that the fetus did inherit the mutated X
chromosome of the maternal subject can be made. The classification
could be confirmed, if necessary, by a second maternal plasma
sample taken at a later stage of pregnancy when fetal DNA
percentages are higher (Lun F M F et al., Clin Chem., 54:1664-1672
(2008)), allowing for more robust testing.
II. CLASSIFICATION BETWEEN NORMAL AND MUTANT
[0052] The analysis in step 155 of method 100 analyzes DNA
fragments in the maternal sample. As the maternal sample also
contains fetal DNA, a genotype of the X chromosome of the male
fetus can be determined. For any mutation on chromosome X, there is
always an allelic imbalance between the concentrations of the
mutant and the wild-type alleles in the plasma of heterozygous
women carrying male fetuses. The overrepresented allele is the one
inherited by the fetus. In one embodiment, the genotype of the
fetus can be determined by the RMD technique, which can include
comparing a number of mutant alleles to a number of normal alleles
in the maternal sample.
[0053] FIG. 2A illustrates the two possibilities of the fetus
inheriting the mutant allele or the normal allele. The maternal DNA
210 is shown for a particular locus on the X chromosomes. The locus
215 is heterozygous with one allele being normal N (wild type) and
the other allele being mutant M. The mutation can be of various
types, such as a different sequence, a deletion, an insertion, and
an inversion. Each of these mutations can be identified as a
different allele than the normal allele at locus 215.
[0054] The fetal DNA 220 is shown with the two possibilities. Since
the male fetus has only one X chromosome, only one of the X
chromosomes of maternal DNA 210 will be inherited by the male
fetus. Possibility 222 shows the male fetus inheriting the mutant
allele M. Possibility 224 shows the male fetus inheriting the
normal allele N. The Y chromosome, which is smaller than the X
chromosome, is also shown for each possibility.
[0055] The maternal sample (e.g. plasma) 230 will have a different
proportion of mutant alleles to normal alleles depending on whether
the fetus inherits the mutant or normal alleles. For possibility
222, the maternal sample will have more mutant alleles M since the
male fetus had inherited the mutant allele M. This is because the
fetal DNA would only contribute the mutant allele M, while the
maternal DNA would contribute roughly equal parts of mutant allele
M and normal allele N when a statistically significant amount of
DNA is analyzed. For possibility 224, the maternal sample will have
more normal alleles N since the male fetus had inherited the normal
allele N.
[0056] The number of DNA fragments showing the normal and mutant
alleles can be counted in various ways, such as digital PCR,
sequencing (including Sanger sequencing, massively parallel
sequencing and single molecule sequencing), and other methods that
would allow the analysis of single DNA molecules or amplified
groups of DNA molecules (e.g. clusters on a solid surface). Once
the number of N and M alleles are counted, various techniques can
be used to perform a classification, such as affected or unaffected
(e.g. a diagnosis of whether the fetus has hemophilia or is
normal). For instance, a parameter (e.g. a ratio or a difference)
can be determined from the number of N and M alleles, and the
parameter can be compared against one or more cutoff values. The
cutoff value(s) can be obtained through various statistical
techniques, such as sequential probability ratio test (SPRT) (Zhou
W, Galizia G, Lieto E, et al., Nat Biotechnol., 19:78-81 (2001);
Zhou W, Goodman S N, Galizia G, et al., Lancet., 359:219-225
(2002)).
[0057] FIG. 2B shows a plot 250 of cutoff values for classifying a
sample as obtained using SPRT according to embodiments of the
present invention. The Y-axis shows the proportion P.sub.r (an
example of a parameter) of alleles that are mutant. The X-axis
shows the number of alleles for locus 215 that are counted. The two
curves correspond to the cutoff values for determining whether the
fetus has the mutation (e.g. hemophilia), is normal, or is
unclassifiable. Samples with mutant allele proportion (P.sub.r)
above the upper boundary and below the lower boundary are
classified as mutant and wild-type, respectively. Samples with
P.sub.r in between the two curves are unclassifiable and require
additional digital analysis (e.g., data from additional PCR
wells).
[0058] The particular cutoff values to use depends on the number of
alleles counted. When only a few alleles are counted, there can be
a large statistical variation, and thus the cutoff values require
extreme values in P.sub.r to confidently classify the sample as
mutant or normal. As is described in more detail below, digital PCR
may be used (where the Y-axis can be the proportion of positive
wells containing the mutant allele and the X-axis can be the number
of positive wells). The position of the curves on the Y-axis can
change depending on how the parameter is calculated, e.g., the
unclassifiable area could be centered at 1.0 if the parameter was
the number of N alleles divided by the number of M alleles.
[0059] In another implementation, where the mutation is a deletion
or amplification, a comparison between a number of fragment at a
target region (e.g. locus 215) where one of the maternal X
chromosomes has a deletion/amplification and a reference region
(not having an amplification or deletion) can be used to identify
the deletion/amplification. Such an implementation does not depend
on an identification of a heterozygous locus, thus the pregnant
subject can be homozygous at the target region. For a deletion, one
would expect fewer fragments from the target region than from the
reference region. For an amplification, one would expect more
fragments from the target region than from the reference region.
The cutoff values can also be determined using SPRT or similar
techniques.
III. RMD METHOD
[0060] FIG. 3 is a flowchart illustrating a method 300 for
determining whether a male fetus of a pregnant female has an
X-linked mutation according to embodiments of the present
invention. The pregnant female is heterozygous for a mutant and a
normal allele at a locus on the X chromosome. Method 300 uses a
relative amount of the mutant and normal allele to make a disease
classification.
[0061] In step 310, data from a plurality of reactions is received.
Each reaction involves one or more nucleic acid molecules from a
biological sample, which includes nucleic acid molecules from the
pregnant female and from the male fetus. The reactions can be of
various types, such as digital PCR reactions in various wells.
Other embodiments can use other reactions, such as sequencing
reactions (for example by a massively parallel sequencing platform,
including but not limited to the Illumina Genome Analyzer, Roche
454, Life Technologies SOLiD, Pacific Biosciences single molecule
real-time sequencing or Ion Torrent), primer extension reactions,
mass spectrometry, analysis using a nanopore, optical methods or
hybridization to a fluorescent or other probe. Thus, the data can
include fluorescent signals from digital PCR wells, sequenced tags
obtained from sequencing at least a portion of the DNA molecules in
the wells, or other data resulting from such reactions.
[0062] The data from the reactions includes a first set of
quantitative data indicating a first amount of the mutant allele at
the locus, and a second set of quantitative data indicating a
second amount of the normal allele at the locus. The amount for a
particular allele at the locus can be measured in various ways,
such as by a total number of wells that are positive for a
particular allele, counting the number of sequenced tags that
include the particular allele and align to the locus (using a
reference genome), and the number of sequenced nucleotides
(basepairs) or the accumulated lengths of sequenced nucleotides
(basepairs) that include the particular allele and align to the
locus.
[0063] In step 320, a parameter is determined from the first amount
and the second amount. The parameter represents a relative amount
between the first and second amounts. The parameter may be, for
example, a simple ratio of the first amount to the second amount,
or the first amount to the second amount plus the first amount. In
one aspect, each amount could be an argument to a function or
separate functions, where a ratio may be then taken of these
separate functions. One skilled in the art will appreciate the
number of different suitable parameters. For example, the parameter
can be a ratio of the number of mutant alleles to the total number
of mutant and wild-type alleles, denoted by P.sub.r, present in a
plasma sample.
[0064] In step 330, a percentage Pf of fetal nucleic acid molecules
in the biological sample is obtained. The percentage Pf provides a
measurement of how much fetal DNA is in the maternal sample
relative to the maternal DNA. If the percentage Pf is higher, then
the overrepresentation of the inherited allele will become larger.
The percentage can be expressed as a fraction between 0 and 1, with
1 being 100%.
[0065] In step 340, a first cutoff value for determining whether
the fetus has inherited the mutant allele at the locus is
calculated. The first cutoff value is derived at least from a first
proportion of 1/(2-Pf). Depending on how the parameter from step
320 is formulated, the proportion 1/(2-Pf) can be equal to the
expected ratio of the first and second amounts if the mutant allele
was inherited. The expected value can be input into a statistical
function to determine the cutoff. The cutoff value may be
determined using many different types of methods, such as SPRT,
false discovery, confidence interval, and receiver operating
characteristic (ROC) curve analysis.
[0066] In step 350, a second cutoff value for determining whether
the fetus has inherited the normal allele at the locus is
calculated. The second cutoff value is derived at least from a
second proportion of (1-Pf)/(2-Pf).
[0067] In step 360, the parameter is compared to at least one of
the first and second cutoff values to determine a classification of
whether the fetus has inherited the mutant allele or the normal
allele. As mentioned above, the classifications can include
affected (mutation inherited) and unaffected (normal inherited),
and also may include unclassified. A probability of accuracy may
also be included with the classification, e.g., the accuracy may be
determined by how much the parameter exceeds (above or below) a
cutoff. In one implementation, the classification may be a score
that is to be interpreted at a later date, for example, by a
doctor.
[0068] The data that indicates an amount of an allele can be from a
linked allele. Thus, an allele that is linked to either the mutant
or the normal allele can be used instead of the normal and mutant
alleles. For example, an allele at a polymorphic site linked to the
mutant nucleic acid sequence can be an allele located on the same
maternal haplotype as the mutant nucleic acid sequence, where the
probability of recombination between the polymorphic site and the
mutant nucleic acid sequence is less than a certain value, e.g. 1%.
Thus, the polymorphic site can provide the same or similar
quantitative data as measuring the mutant allele directly. As
another example, an allele at a polymorphic site linked to the
normal nucleic acid sequence can be an allele located on the same
maternal haplotype as the normal nucleic acid sequence, where the
probability of recombination between the polymorphic site and the
mutant nucleic acid sequence is less than a certain value, e.g.
1%.
[0069] A. Example Using PCR with Plasma
[0070] As mentioned above, digital PCR can be used as the method
for identifying DNA fragments that include the mutant or normal
allele. In digital PCR, a sample is separated into a plurality of
compartments (e.g., wells and beads). On average, each compartment
contains less than one of any of the two alleles. Thus, a positive
well can be counted as a single instance of a fragment containing
the allele.
[0071] FIG. 4 illustrates a method 400 for determining whether a
male fetus has inherited an X-linked mutation according to
embodiments of the present invention. Digital PCR is used to
determine a mutant allele proportion and the fetal DNA percentage.
The fetal DNA percentage is used to determine a cutoff value to
which the mutant allele proportion is compared, thereby providing a
classification of whether the male fetus has inherited the
mutation. As the mutant allele proportion is determined,
embodiments can be referred to as the RMD method.
[0072] As illustrated, for each maternal plasma DNA sample, both
the mutant DNA proportion (P.sub.r) and the fetal DNA percentage Pf
are determined by digital PCR, although other reactions that can
identify certain sequences may be used. Steps for determining
P.sub.r is provided on the left (process 401), and steps for
determining the fractional fetal DNA concentration Pf are on the
right (process 402). As shown, P.sub.r is determined using a
real-time PCR assay targeting the mutation carried by the mother,
while the fetal DNA percentage Pf is determined using the real-time
PCR assay for the homologous ZFY and ZFX gene regions.
[0073] In step 410, the PCR mixture is prepared. As shown, the
mixtures are different for the two measurements. For the P.sub.r
measurement (process 401), the mixture contains PCR primers to
amplify a region on the X chromosome that includes the locus to be
tested. The mixture also contains a fluorescent probe to identify
the existence of a DNA fragment with the wild-type allele, and a
fluorescent probe to identify the existence of a DNA fragment with
the mutant allele. For the Pf measurement (process 402), the
mixture contains primers for the ZFY and ZFX gene regions. The
mixture also includes fluorescent probe to identify the existence
of a DNA fragment containing a sequence from the ZFX gene, and a
fluorescent probe to identify the existence of a DNA fragment
containing a sequence from the ZFY gene.
[0074] In step 420, the reaction mixtures are loaded into a PCR
machine. In one embodiment, the digital PCR is carried out in a
microfluidics Digital Array (Fluidigm), which consists of 12 panels
with each panel further partitioned into 765 reaction chambers.
Each DNA sample (i.e. one for P.sub.r and one for Pf) is analyzed
using 6 panels, i.e., 765.times.6=4590 chambers. The PCR mixture
can be first manually added into the sample inlet of each panel.
The mixture is next aliquoted into 765 chambers in each panel
automatically by an Integrated Microfluidics Circuit Controller
(Fluidigm). Each chamber contains a final reaction volume of 6 nL.
The cell-free DNA concentration in maternal plasma is typically
very low such that there is less than one template molecule per
chamber on average. Hence, the distribution of template molecules
to the chambers follows the Poisson distribution. For other
samples, one may need to dilute the DNA sample before analysis. It
will also be obvious to those of skill in the art that the digital
PCR can be performed using methods well-known to those of skill in
the art, e.g. microfluidics chips, nanoliter PCR microplate
systems, emulsion PCR (including the RainDance platform), polony
PCR, rolling-circle amplification, primer extension and mass
spectrometry.
[0075] As shown for the P.sub.r measurement, wells (chambers)
containing a DNA fragment with the wild-type allele are shown in
blue, and wells containing a DNA fragment with the mutant allele
are shown in red. Wells that do not contain a temple DNA molecule
(i.e. no allele for which there is a probe) are shown simply as
white. Similarly for the Pf measurement, wells containing the ZFX
gene are shown in blue, and wells containing the ZFY gene are shown
in red.
[0076] In step 430, real-time PCR is performed, e.g., on the
BioMark System (Fluidigm). Each well is carried through a series of
cycles that amplify DNA regions that correspond to the primers in
the corresponding mixture. Since most of the chambers contain zero
or one template DNA molecule, the amplified products from a well
originate from one template DNA molecule.
[0077] In step 440, the number of chambers with positive PCR
amplifications are counted. For the process 401, the number of
chambers that are positive for the wild-type allele can be counted
and the number of chambers for the mutant allele can be counted.
For process 402, the number of chambers that are positive for the
ZFX gene can be counted and the number of chambers for the ZFY gene
can be counted. In each process, the number of chambers that are
positive for both of the alleles can also be identified. The
detection of a positive chamber can be performed in various ways,
such as detecting a fluorescent signal (e.g. each allele will emit
a different color signal). For example, chambers containing the ZFX
gene can emit a blue fluorescent signal, and wells containing the
ZFY gene can emit a red fluorescent signal.
[0078] In step 450, the mutant DNA proportion (P.sub.r) and the
fetal DNA percentage Pf are calculated using the corresponding
numbers counted in step 440. For example, the mutant allele
proportion could be calculated as the number of chambers positive
for the mutant allele divided by the total number of positive
wells. As other examples, the denominator could be the total number
of chambers that are positive only for one allele. Instead of a
ratio involving the raw number of counts, the values could be
concentrations themselves, effectively dividing the numerator and
the denominator by any of the values above. Similar values can be
used to calculate the fetal DNA percentage Pf using the equation
[(2Y)/(X+Y)]*100%, where Y is the measured amount for the ZFY gene
(e.g., count of positive chambers or proportion of positive
chambers), and X is the measured amount for the ZFX gene.
[0079] Since there was less than one template molecule per reaction
well, the actual number of template molecules distributed to each
reaction chamber followed the Poisson distribution. Hence, the
number of chambers for any allele can be Poisson-corrected using
the equation [-ln((N-P)/N)]*N, where N is the total number of
reaction chambers analyzed, P is the number of chambers positive
for the allele, and ln is the natural logarithm. The
Poisson-corrected values can then be used in a similar manner as
mentioned above to determine the proportion P.sub.r and the fetal
DNA percentage Pf.
[0080] In step 460, the mutant DNA proportion (P.sub.r) and the
fetal DNA percentage Pf are used to perform a classification of
whether the male fetus had inherited the mutation or not. As for
method 300, cutoff values can be determined from the fetal DNA
percentage Pf, e.g., as in steps 340 and 350. The cutoff may also
be derived from (which includes equal to) an average reference
template concentration (m.sub.r), e.g., the experimentally measured
percentage of positive chambers for the wild-type allele can be
used to determine the cutoff value used in step 460. This strategy
can further minimize the amount of testing required before
confident classification could be made. This is of particular
relevance to plasma nucleic acid analysis where the template amount
is often limiting.
[0081] B. SPRT
[0082] SPRT is a method which allows two probabilistic hypotheses
to be compared as data accumulate. In other words, it is a
statistical method to classify the results of digital PCR as being
suggestive of the skewing towards either the mutant or the normal
allele. It has the advantage of minimizing the number of wells to
be analyzed to achieve a given statistical power and accuracy.
[0083] In an exemplary SPRT analysis, the experimental results
would be tested against two alternative hypotheses. The first
alternative hypothesis is accepted when the mutant allele is
over-represented. The second alternative hypothesis is accepted
when the mutant allele is under-represented. The measured P.sub.r
would be compared with at least one of the two cutoff values to
accept the first or the second alternative hypotheses. If neither
hypothesis is accepted, the sample would be marked as unclassified
which means that the observed digital PCR result is not sufficient
to classify the sample with the desired statistical confidence.
More data can be collected to obtain the desired statistical
confidence.
[0084] A pair of curves, which depend on the amount of data
collected, can define the probabilistic boundaries (cutoffs) for
accepting or rejecting the hypotheses (Zhou W, Galizia G, Lieto E,
et al., Nat Biotechnol., 19:78-81 (2001); Zhou W, Goodman S N,
Galizia G, et al., Lancet., 359:219-225 (2002)). The SPRT curves
delineated the required P.sub.r (y-axis) for a given total number
of positive reactions (x-axis) for classifying a fetal genotype.
Hypothesis (i) or (ii) are accepted if the experimental P.sub.r
fell above the upper boundary or below the lower boundary,
respectively. The equations for calculating the SPRT boundaries can
be determined with varying levels of statistical confidence (e.g.
adjusted to a threshold likelihood ratio of 8). In one aspect, the
cutoff values of the SPRT curves are sample-specific. The cutoff
values are dependent on the fractional fetal DNA concentration
(fetal DNA percentage) as described above. The cutoff values can
also depend on an average reference template concentration per PCR
well (m.sub.r) for a given set of reactions (Lo Y M D et al., Proc
Natl Acad Sci USA. 2007; 104:13116-13121 (2007); Lun F M F, Tsui N
B Y, Chan K C A, et al., Proc Natl Acad Sci USA.,105:19920-19925
(2008)). The reference template can refer to the allele that showed
the lesser positive amplification counts in the sample.
[0085] SPRT can offer an advantage that a smaller amount of testing
is required for a given level of confidence than other statistical
methods. In practical terms, SPRT allows the acceptance or
rejection of either of the hypotheses as soon as the required
amount of data has been accumulated and thus minimizes unnecessary
additional analyses. This feature is of particular relevance to the
analysis of plasma nucleic acids which are generally present at low
concentrations where the number of available template molecules is
limiting. In addition to a strict classification, the
classification may also include a percent accuracy. For example, a
classification resulting from a comparison with a cutoff value may
provide that a sample shows a likelihood of a nucleic acid sequence
imbalance with a certain percentage, or equivalently that a
determined imbalance is accurate to a certain percentage or other
value.
[0086] For embodiments using SPRT, one may use the equations for
calculating the upper and lower boundaries of the SPRT curves from
El Karoui at al (El Karoui N, Zhou W, Whittemore A S, Stat Med.
25:3124-3133 (2006)). Furthermore, the level of statistical
confidence preferred for accepting the first or second hypothesis
could be varied through adjusting the threshold likelihood ratio in
the equations. A threshold likelihood ratio of 8 has been shown to
provide satisfactory performance to discriminate samples with and
without allelic imbalance in the context of cancer detection. Thus,
in one embodiment, the equations for calculating the upper and
lower boundaries of the SPRT curves are:
Upper boundary=[(ln 8)/N-ln .delta.]/ln .gamma.
Lower boundary=[(ln 1/8)/N-ln .delta.]/ln .gamma.
where, .delta.=(1-0.sub.1)/(1-0.sub.2),
.gamma. = .theta. 1 ( 1 - .theta. 2 ) .theta. 2 ( 1 - .theta. 1 ) ,
##EQU00001##
ln is a mathematical symbol representing the natural logarithm,
i.e. log.sub.e, N=total number of molecules (i.e. the sum of mutant
and normal molecules analyzed), [0087] .theta..sub.1=proportion of
mutant molecules to the total number of mutant and normal molecules
if the first alternative hypothesis is true (i.e., the fetus has
inherited the mutant allele); and [0088] .theta..sub.2=proportion
of mutant molecules to the total number of mutant and wild-type
molecules if the second alternative hypothesis is true (i.e., the
fetus has inherited the normal allele).
[0089] For the determination of .theta..sub.1 for accepting the
first alternative hypothesis, the sample is assumed to be obtained
from a pregnant woman carrying a male fetus which has inherited the
mutant (M) allele. .theta..sub.1 is determined to be 1/(2-Pf),
where Pf is the percentage of fetal DNA in the sample. Pf can be
corrected for a statistical distribution, such as the Poisson
distribution, as is described herein.
[0090] For the determination of .theta..sub.2 for accepting the
second alternative hypothesis, the sample is assumed to be obtained
from a pregnant woman carrying a male fetus which has inherited the
normal (N) allele. .theta..sub.2 is determined to be
(1-Pf)/(2-Pf).
[0091] After an experimental determination of the numbers of mutant
and wild-type molecules, the proportion of mutant molecules to the
total number of mutant and wild-type molecules (Pr) can be
calculated. The value of Pr can then be compared with the cutoff
values to determine if the mutant or the wild-type alleles are
overrepresented in the maternal plasma.
[0092] C. Poisson Correction of Cutoff Values
[0093] In one embodiment using digital PCR, the average
concentration per well (reaction or reaction mixture) is
determined, and the expected number of wells showing that sequence
may be calculated. This amount may be expressed as a percentage, a
fractional value, or an integer value. In one implementation, a
Poisson distribution is assumed for the distribution of the normal
(N) allele, or the mutant allele, among the reaction mixtures of
the wells of the measurement procedure, such as digital PCR. In
other implementations, other distribution functions are used, such
as a binomial distribution.
[0094] The Poisson equation is:
P ( n ) = m n e - m n ! ##EQU00002##
where, n=number of template molecules per well; P(n)=probability of
n template molecules in a particular well; and m=average number of
template molecules in one well in a particular digital PCR
experiment. Accordingly, the probability of any well not containing
any molecule of the normal allele at an average normal-allele
concentration of 0.5 would be:
P ( 0 ) = 0.5 0 e - 0.5 0 ! = e - 0.5 = 0.6065 . ##EQU00003##
[0095] Hence, the probability of any well containing at least one
molecule of the normal allele would be: 1-0.6065=0.3935. Therefore,
.about.39% of the wells would be expected to contain at least one
molecule of the normal allele. In one embodiment, P(0) for mutant
or wild-type can be determined from an experimentally derived
proportion of negative wells (e.g. using digital PCR). P(0) can
then be used to calculate the average number of molecules per well
(m). The parameter can then be calculated from the average number
of molecules per well, e.g., mutant average divided by the sum of
the averages for the mutant and normal alleles. Given this
relationship between the number of positive wells and the number of
molecules, an alternative is to correct the number of positive
wells to provide the number of molecules (as described above via
equation [-ln((N-P)/N)]*N, where N is the total number of reaction
chambers analyzed and P is the number of chambers positive for the
allele).
[0096] The measurement of m.sub.r may be performed through a
variety of mechanisms as known or will be known to one skilled in
the art. In one embodiment, the value of m.sub.r is determined
during the experimental process of digital PCR analysis. As the
relationship between the value of m.sub.r and the total number of
wells being positive for the reference allele can be governed by a
distribution (e.g. the Poisson distribution), m.sub.r can be
calculated from the number of wells being positive for the
reference allele using this formula:
m.sub.r=-ln (1-proportion of wells being positive for the reference
allele)
This approach provides a direct and precise estimation of m.sub.r
in the DNA sample used for the digital PCR experiment.
[0097] This method may be used to achieve a desired concentration.
For example, the extracted nucleic acids of a sample may be diluted
to a specific concentration, such as one template molecule per
reaction well. In an embodiment using the Poisson distribution, the
expected proportion of wells with no template may be calculated as
e.sup.-m, where m is the average concentration of template
molecules per well. For example, at an average concentration of one
template molecule per well, the expected proportion of wells with
no template molecule is given by e.sup.-1, i.e., 0.37 (37%). The
remaining 63% of wells will contain one or more template molecules.
Typically, the number of positive wells in a digital PCR run would
then be counted. The definition of informative wells and the manner
by which the digital PCR data are interpreted depends on the
application.
[0098] In other embodiments, the average concentration per well,
m.sub.r, is measured by another quantification method, for example,
quantitative real-time PCR, semi-quantitative competitive PCR, and
real-competitive PCR using mass spectrometric methods.
[0099] In one implementation, the proportion of the mutant allele
to the normal allele can be calculated using corrected
concentrations. The concentration m for each allele can be
calculated as described above. The concentration for each allele
can then be determined, and a proportion Pr of the concentrations
can be used as the experimentally derived and
distribution-corrected proportion to compare to the expected
proportion for each hypothesis (e.g. mutant or wild-type
inheritance). For example, the experimentally determined Pr of a
tested sample can be calculated using the equation: (concentration
of mutant allele)/(concentration of mutant+wild-type alleles). In
another implementation, the proportion of the number of wells for
each allele is used. The expected proportion (cutoff value) can
also be corrected based on a statistical distribution.
[0100] D. Illustration
[0101] FIG. 5A shows a table 500 illustrating a dosage imbalance
between mutant and wild-type alleles for mutations on chromosome X
according to embodiments of the present invention. To illustrate
the calculation, a maternal plasma sample containing a total of 100
genomic equivalents (GE) of DNA with 10% fetal DNA was used. For
the maternal genome, one GE contains two copies of the alleles,
i.e., one copy each of the M and the N allele. This provides 90
copies each of the mutant and normal alleles. For the fetal genome,
one GE contains one copy of the X-linked allele, i.e., one copy of
either the mutant (M) or the normal (N) allele. This provides 0 or
10 copies of each allele depending on which allele is inherited by
the fetus.
[0102] In table 500, the upper row corresponds to the fetus
inheriting the normal allele, and thus the ratio of mutant to
normal alleles is less than 1. In the lower row, the fetus
inherited the mutant allele, and thus the ratio of mutant to normal
alleles is greater than 1.
[0103] E. Deletions, Amplifications, Insertions, and Inversions
[0104] Methods 300 and 400 can be applied in additional situations
besides a standard SNP. Embodiment can be further applied to
noninvasive detection of fetal mutations involving deletion,
amplification (e.g. duplication), insertion, and inversion, e.g.,
of a large DNA segment. Examples of such mutations are relevant to
X-linked diseases such as Duchenne muscular dystrophy, Becker
muscular dystrophy and ornithine transcarbamylase deficiency. The
approach is to detect the mutant allele by targeting the junctions
of the rejoining sequences of the deletion, between the amplified
(e.g. duplicated) DNA segments, or between the inverted and the
adjacent normal DNA segments. The fetal genotype could then be
deduced by the dosage imbalance between the normal and the mutant
alleles with the methods described herein.
[0105] FIG. 5B illustrates a first scenario for detecting an
amplification when the pregnant subject is heterozygous at the
locus of interest. For amplifications on a first chromosome, where
the amplified allele B is different than the non-amplified allele
A, there will be different junctions for the various copies B1 and
B2 of the amplified allele B. This is because the amplified copies
B1 and B2 will be at different locations on the first chromosome.
If one of the junctions is unique (e.g., the junction at the start
of B or at the end of B2 is unique, while the junctions between
B-B1 and B1-B2 are the same), the unique junction can be used as
the mutant allele for comparison to the normal allele on the other
chromosome. In this manner, the cutoff values can be derived in the
same manner as in steps 340 and 350. Alternatively, all of the
instances of the amplified allele B (i.e. is B, B1, and B2) can be
used, regardless of location in the first chromosome. In such an
embodiment, .theta..sub.1=(1+n)/(2+n-Pf), and
.theta..sub.2=[(1+n)(1-Pf)]/[2+n-Pf(1+n)], where n is the number of
additional copies (n=2 as shown), where n is an integer equal to or
greater than zero. These formulas can also be written as
.theta..sub.1=k/(1+k-Pf) and .theta..sub.2=[k(1-Pf)]/[1+k-kPf)],
where k is the number of copies of the mutant allele (which can be
a newly formed junction) on the mutant chromosome, where k is an
integer equal to or greater than one.
[0106] Junctions can also be used in a similar manner for RMD
analysis for mutations on autosomes, but the values of
.theta..sub.1 and .theta..sub.2 would need to be adjusted. For
example, if the fetus inherited the amplification mutation, the
sample would have the same ratio as the mother, assuming the
chromosome inherited from the father is the normal chromosome. In
this scenario, the value of .theta..sub.1 would be k/(k+1), where k
is the number of additional junctions created by the amplification
mutation, and the additional junction is used as the mutant allele
(thus for a duplication or a deletion, there is one mutant allele
and for a triple amplification there are two mutant alleles, and so
on). If the fetus inherited the normal chromosome from the mother,
then the value of .theta..sub.2 would be k(1-Pf)/[k+1+(1-k)Pf].
[0107] FIG. 5C illustrates a second scenario for detecting an
amplification when the pregnant subject is homozygous at the locus
of interest. When the amplified allele and the non-amplified allele
are the same (A as shown), two junctions 510 will be the same (for
the two alleles at the normal location), and the additional (new)
junction(s) 520 of the additional copies of the allele will be
different, since these additional alleles will be at a different
genomic location. The additional junctions can be used as the
mutant allele, and the normal junction 510 can be used as the
normal allele. One can use just one of the additional junctions 520
for the additional allele(s) (there would be only one for a
duplication). In such an embodiment, .theta..sub.1=1/(3-Pf); and
.theta..sub.2=(1-Pf)/(3-2Pf). Note that the amount of additional
copies is not used in such formulas since just one additional
junction is used.
[0108] If there are more than one additional copy of A, the
additional junction that is used should be chosen to be unique
(e.g. the junction after the last amplified copy of A). Or, one
could sum all (or some number more than 1) of the additional
junctions and compare to the junctions of the two alleles at the
normal location. In such an embodiment, .theta..sub.1=n/(n+2-Pf);
and .theta..sub.2=n(1-Pf)/[n+2-Pf(n+1)], where n is the number of
new junctions 520 that are used. Note that the amount of additional
copies is used in such formulas since just more than one additional
junction is used. Junctions can also be used in a similar manner
for RMD analysis for mutations on autosomes, but the values of
.theta..sub.1 and .theta..sub.2 would need to be adjusted. For
example, if the fetus inherited the amplification mutation
(amplification), the sample would have the same ratio (e.g., 1:2
for a duplication) as the mother, assuming the chromosome inherited
from the father does not have the mutation. In this scenario, the
value of .theta..sub.1 would be n/(n+2), where n is the number of
additional junctions created by the amplification mutation. If the
fetus inherited the normal chromosome from the mother, then the
value .theta..sub.2 would be n(1-Pf)/(n+2-nPf). Another approach
for detecting deletions and amplifications is described below.
IV. TARGET REGION VS REFERENCE REGION
[0109] In the RMD method described above, different junctions can
be used as the alleles when the mutation is a deletion,
amplification, insertions, or inversion. Another approach, which is
applicable to deletion and amplification (e.g. duplication)
mutations, is to compare the amount of molecules arising from the
target region (i.e. the region that is deleted or amplified) to the
amount of molecules arising from a reference region. Any genomic
locus on chromosome X not affected by the deletion (or
amplification) can be used as a reference locus/region, for
example, the ZFX gene if it is not deleted or amplified.
[0110] The ratio (R) of the number of molecules from the target
region to the number of molecules from the reference region (or
some other parameter representing a relative amount) can be used to
determine whether the mutation is inherited. In a non-pregnant
woman who is carrying the deletion mutation, the expected value of
R would be 0.5 because only half of the X chromosomes (those
carrying the normal allele) would contribute to the amount of
target molecules in the plasma. When a woman carrying this deletion
mutation is pregnant with a male fetus, the expected value of R
would deviate from 0.5 due to the contribution of the DNA from the
one extra X chromosome from the male fetus. The expected deviation
of R would depend on whether the mutation is a deletion or an
amplification.
[0111] FIG. 6 is a flowchart illustrating a method 600 for
determining whether a male fetus of a pregnant female has an
X-linked mutation. The pregnant female is heterozygous for a
mutation and a normal allele at a target region on the X
chromosome. The mutation is a deletion or an amplification of the
target region.
[0112] In step 610, data from a plurality of reactions is received.
The data may be of the same type as received in step 310 of method
300. Each reaction involves one or more nucleic acid molecules from
a biological sample, which includes nucleic acid molecules from the
pregnant female and from the male fetus. The data includes a first
set of quantitative data indicating a first amount of the nucleic
acid molecules that are from the target region, and a second set of
quantitative data indicating a second amount of the nucleic acid
molecules that are from a reference region on the X chromosome. The
amounts may be computed in various ways, e.g., as described above
for step 310.
[0113] In step 620, a parameter is determined from the first amount
and the second amount. The parameter represents a relative amount
between the first and second amounts. In one embodiment, the
parameter is a ratio T of the first amount to the second amount.
Other embodiments can use parameters as described herein, such the
first amount divided by a sum of the first amount and the second
amount.
[0114] In step 630, a percentage Pf of fetal nucleic acid molecules
in the biological sample is obtained. The percentage Pf can be
calculated as described herein. The percentage Pf can also be
determined from a distribution corrected (e.g. Poisson-corrected)
values for counting fetal specific molecules.
[0115] In step 640, a first cutoff value for determining whether
the fetus has inherited the mutation is calculated. The first
cutoff value is dependent on the percentage Pf. The specific
equations for calculating the first cutoff value depends on whether
the mutation is a deletion or an amplification.
[0116] In step 650, a second cutoff value for determining whether
the fetus has inherited the normal allele is calculated. The second
cutoff value is dependent on the percentage Pf. The specific
equations for calculating the first cutoff value depends on whether
the mutation is a deletion or an amplification.
[0117] In step 660, the parameter is compared to at least one of
the first and second cutoff values to determine a classification of
whether the fetus has inherited the mutant or the normal allele.
The classifications can be of the same type as step 360, such as
affected, unaffected, or unclassified (or a raw score).
[0118] FIG. 7 is a table 700 showing a dosage imbalance between the
target and the reference loci for deletion and duplication
mutations on chromosome X. Table 700 illustrates the calculation of
the degree of allelic imbalance. An increase or decrease of R when
compared with R of a non-pregnant woman carrying the same deletion
mutation would indicate a normal or affected fetus, respectively.
Conversely, in a non-pregnant woman who is carrying the segmental
amplification, such as a duplication as shown in table 700, the
expected value of R would be 1.5 due to the contribution of a
doubled dose of target molecules from the mutant allele. When a
woman carrying this duplication mutation is pregnant, an increase
or decrease of R when compared with R of a non-pregnant woman
carrying the same duplication mutation would indicate an affected
or normal fetus, respectively.
[0119] The degree of increase or decrease of R in each scenario is
dependent on the fractional fetal DNA concentration (Pf) in a
sample. In one embodiment, SPRT analysis can be used to determine
if R is statistically significantly increased or decreased compared
to the non-pregnant women carrying the same mutation. The equations
for calculating the upper and lower boundaries (cutoff values) of
the SPRT can have a similar structure of:
Upper boundary=[(ln 8)/N-ln .delta.]/ln .gamma.;
Lower boundary=[(ln 1/8)/N-ln .delta.]/ln .gamma.
where .delta.=(1-.theta..sub.1)/(1-.theta..sub.2);
.gamma. = .theta. 1 ( 1 - .theta. 2 ) .theta. 2 ( 1 - .theta. 1 ) ;
##EQU00004##
ln is a mathematical symbol representing the natural logarithm,
i.e. log.sub.e; N=total number of mutant and reference molecules;
[0120] .theta..sub.1=ratio (R.sub.1) of target molecules to the
reference molecules if the first alternative hypothesis is true
(i.e., R.sub.1 is increased when compared with the value of R of a
non-pregnant woman carrying the same mutation) [0121]
.theta..sub.2=ratio (R.sub.2) of target molecules to reference
molecules if the second alternative hypothesis is true (i.e.,
R.sub.2 is decreased when compared with the value of R of a
non-pregnant woman carrying the same mutation)
[0122] .theta..sub.1 describes the situation in which the ratio of
the amount of target molecules to the amount of reference molecules
is increased when compared to the corresponding ratio of a
non-pregnant woman carrying the same mutation, e.g., a normal case
for a deletion mutation, or a mutant case for a duplication
mutation. Similarly, .theta..sub.2 can describe the situation in
which the ratio of the amount of target molecules to the amount of
reference molecules is decreased when compared to the corresponding
ratio from a non-pregnant woman carrying the same mutation, e.g., a
mutant case for a deletion mutation, or a normal case for a
duplication mutation.
[0123] In one embodiment, for a deletion mutation, .theta..sub.1 is
calculated as the sample is assumed to be obtained from a pregnant
woman carrying a male fetus that has inherited the normal (N)
allele. .theta..sub.1 is determined to be 1/(2-Pf). .theta..sub.2
is calculated as the sample is assumed to be obtained from a
pregnant woman carrying a male fetus that has inherited the
mutation (e.g. the chromosome X with the deletion mutation).
.theta..sub.2 is determined to be (1-Pf)/(2-Pf).
[0124] In another embodiment, for duplication mutation,
.theta..sub.1 is calculated as the sample is assumed to be obtained
from a pregnant woman carrying a male fetus that has inherited the
mutation (i.e. the chromosome X with the duplication mutation).
.theta..sub.1 is determined to be (3-Pf)/(2-Pf). .theta..sub.2 is
calculated as the sample is assumed to be obtained from a pregnant
woman carrying a male fetus that has inherited the normal (N)
allele. .theta..sub.2 is determined to be (3-2.times.Pf)/(2-Pf).
The generalized formulas for any level of amplification is:
.theta..sub.1 is (n+2-Pf)/(2-Pf), and .theta..sub.2 is
[n+2-Pf(n+1)]/(2-Pf), where n is the number of additional copies of
amplified segments.
V. DETERMINING FETAL PERCENTAGE
[0125] As mentioned above, probabilities P(n) for certain alleles
(e.g. specific to chromosome X and a fetal-specific sequence) can
be used to adjust the percentage (Pf) of fetal DNA in the sample.
This adjusted Pf can then be used to calculate the cutoffs for
determining whether the mutant or the wild-type allele is
inherited.
[0126] FIG. 8 is a flowchart illustrating a method 800 for
obtaining a percentage Pf of fetal nucleic acid molecules in a
biological sample from a female pregnant with a fetus according to
embodiments of the present invention. The biological sample
includes nucleic acid molecules from the pregnant female and from
the fetus.
[0127] In step 810, data is received from a plurality of reactions.
Each reaction involves a plurality of nucleic acid molecules from a
biological sample. In one aspect, the reactions may be of any type
where a reaction is considered positive for a particular allele if
one or more of the alleles are present in the reaction.
[0128] In step 820, a first allele is detected in the reactions.
The first allele is shared by the mother and fetus at a locus where
the pregnant female is homozygous and the fetus is either
heterozygous or hemizygous. In one embodiment, the first allele is
the X chromosome.
[0129] In step 830, a corrected concentration Px of the first
allele is calculated based on a number of reactions positive for
the first allele. Px is corrected for an expected statistical
distribution of the first allele in the plurality of reactions. For
example, Px can be corrected based on the Poisson distribution. In
one embodiment, a first corrected concentration for a first allele
shared by the mother and fetus where the mother is homozygous and
the fetus is either heterozygous or hemizygous is calculated, e.g.,
as [-ln((N-P1)/N)]*N, where N is the total number of reaction
chambers analyzed, P1 is the number of chambers positive for the
first allele, and ln is the natural logarithm.
[0130] In step 840, a second allele that is specific to the fetus
is detected. In one embodiment, the second allele is on the Y
chromosome, where the fetus is a male fetus. In another embodiment,
the fetal-specific allele is a paternally-inherited allele on an
autosome. In yet another embodiment, the fetal-specific allele
includes a methylation marker specific to the fetus.
[0131] In step 850, a corrected concentration Py of the second
allele is calculated based on a number of reactions positive for
the second allele. Py is corrected for an expected statistical
distribution of the second allele in the plurality of reactions.
For example, Py can be corrected based on the Poisson distribution.
In one embodiment, a second corrected concentration for a
fetal-specific allele which the fetus is heterozygous or hemizygous
can be calculated as [-ln((N-P2)/N)]*N, where N is the total number
of reaction chambers analyzed, P2 is the number of chambers
positive for the fetal-specific allele, and ln is the natural
logarithm.
[0132] In step 860, the percentage Pf of fetal nucleic acid
molecules in the biological sample is calculated using
[(2Py)/(Px+Py)], which can provide a fractional value. The fetal
DNA percentage can be calculated using the equation
[(2P2)/(P1+P2)]*100%.
VI. EXAMPLES
[0133] Seven women who were carriers of hemophilia (three carriers
of hemophilia A, four carriers of hemophilia B) and pregnant with
male fetuses were recruited from the Royal Free Hospital, London,
UK. We also recruited 20 pregnant women (non-carriers of
hemophilia) each pregnant with a singleton healthy male fetus. Ten
of them were recruited from the Royal Free Hospital, London, UK and
the other ten were recruited from the Prince of Wales Hospital,
Hong Kong. Clinical information of the cases is shown in table 900
of FIG. 9, which shows clinical information of the seven pregnant
women who are carriers of hemophilia mutations.
[0134] All women were recruited with informed consent. Ethical
approvals were granted by the respective institutional boards. Ten
milliliters of peripheral blood samples was collected into EDTA
tubes from the pregnant women. For five of the pregnant hemophilia
carriers, peripheral blood samples were taken on two occasions
during their pregnancies (table 900). None of the pregnant
hemophilia carriers in this study had invasive prenatal testing.
Fetal sex and hemophilia status were confirmed following delivery.
For the ten unaffected pregnant women recruited in Hong Kong,
placental tissues were also collected following deliveries.
[0135] We centrifuged the blood samples at 1600 g for 10 min at
4.degree. C. The plasma portion was recentrifuged at 16000 g for 10
min at 4.degree. C. Maternal plasma and buffy coat samples were
stored at -20.degree. C. until further processing. All samples
collected in the UK were processed and stored frozen locally and
were shipped on dry ice to Hong Kong. We extracted DNA from
maternal plasma with the QIAamp DSP DNA Blood Mini Kit (Qiagen)
following the manufacturer's instructions. Buffy coat DNA was
extracted using the Illustra DNA Extraction Kit (GE Healthcare)
following the manufacturer's protocol.
Genotyping of rs6528633 SNP and Hemophilia Mutations
[0136] To assess the feasibility of the RMD approach, we studied a
SNP (rs6528633) on chromosome X. This SNP was chosen for
illustration purposes and other SNPs can be used. The fetal and
maternal SNP genotypes were determined using DNA obtained from the
placental and maternal buffy coat samples, respectively. Genotyping
was performed using MassARRAY homogenous MassEXTEND (hME) assays
(Sequenom) as previously described (Tsui N B Y, Chiu R W K, Ding C,
et al., Clin Chem., 51:2358-2362 (2005); Tsui N B Y, Chiu R W K,
Ding C, et al., Clin Chem., 51:2358-2362 (2005)). Genomic DNA
obtained from the peripheral blood samples of the pregnant
hemophilia carriers was used for hemophilia mutation detection.
PCRs were performed for all exons covering coding regions,
intron/exon boundaries, promoter and 3' UTR. Cycle sequencing was
carried out using Big Dye Terminators V1.1 (Applied Biosystems) and
analyzed on an Applied Biosystems 3100 Avant Genetic Analyser.
Digital RMD Reactions for Maternal Plasma Analyses
[0137] The experimental workflow of digital RMD is illustrated in
FIG. 4 according to certain embodiments of the present invention.
We measured the fractional fetal DNA concentrations in the maternal
plasma samples using the previously described digital ZFY/X assay,
which quantified the homologous ZFY and ZFX gene loci located on
chromosomes Y and X, respectively (Lun F M F et al., Clin Chem.,
54:1664-1672 (2008); Lun F M F, Tsui N B Y, Chan K C A, et al.,
Proc Natl Acad Sci USA.,105:19920-19925 (2008)). For the rs6528633
SNP, a real-time PCR assay with two allele-specific TaqMan probes
(Applied Biosystems) was designed to distinguish the two SNP
alleles. For the mutations of the pregnant cases at risk for
hemophilia, a real-time PCR assay for allelic discrimination was
designed for each mutation. Each assay contained two
allele-specific TaqMan probes for the mutant and the wild-type
alleles. The primer and probe sequences are listed in table 1000 in
FIG. 10, which shows oligonucleotide sequences and real-time PCR
conditions for the allele-discriminative assays. In other
emdodiments, the fractional fetal DNA concentration can be
determined by using a sequence that is differentially methylated
between the fetal and maternal DNA in maternal plasma (for
examples, see Chim S S et al., Proc Natl Acad Sci USA., 102:
14753-14758 (2005); Chan K C A et al., Clin Chem., 52: 2211-2218
(2006)).
[0138] We performed digital PCR analyses on the BioMark System
(Fluidigm) using the 12.765 Digital Arrays (Fluidigm) (Lun F M F et
al., Clin Chem., 54:1664-1672 (2008)). Six of the 12 panels on the
Digital Array were used for each DNA sample, which corresponded to
4590 individual PCRs. The reaction for one sample (6 panels) was
set up using 2.times. TaqMan Universal PCR Master Mix (Applied
Biosystems) in a reaction volume of 52 .mu.L. The reactions were
set up according to the manufacturer's protocol with the primer and
probe compositions listed in table 1000 of FIG. 10. Each reaction
mix contained 18.2 .mu.L of the DNA sample. The reaction mixture
was automatically loaded onto the Digital Array by the NanoFlex IFC
Controller (Fluidigm). The reactions were carried out on the
BioMark System (Fluidigm). The reactions were initiated at
50.degree. C. for 2 minutes, followed by 95.degree. C. for 10
minutes, and 45 cycles of 95.degree. C. for 15 seconds and
assay-specific annealing temperatures (FIG. 10 TABLE 3) for 1
minute. For a sample that remained unclassified by the RMD with
data from one 4590-well digital PCR set, additional 4590-well
digital PCR sets were carried out until a genotype call could be
made.
Results
[0139] Principle of Digital RMD for X-Linked Polymorphisms
[0140] Embodiments can use digital PCR to measure the concentration
difference between the total amount (maternal-plus fetal-derived)
of mutant and wild-type alleles in the plasma of heterozygous
pregnant women carrying male fetuses. Since a male fetus possesses
a single chromosome X, the relative concentration between the
wild-type and the mutant allele is always in dosage imbalance (FIG.
2A). An over- or under-representation of the mutant allele
represents an affected or normal fetus, respectively. We used SPRT
to test for dosage imbalance. A pair of SPRT curves was constructed
(FIG. 2B). Samples with data points above the upper curve or below
the lower curve were classified as affected or normal,
respectively. Samples with data points in between the two curves
were not classified because of insufficient statistical power and
additional digital PCRs would be performed.
[0141] Noninvasive Determination of the Fetal Genotype for a SNP on
Chromosome X
[0142] We used a SNP, rs6528633 (A/T polymorphism), on chromosome X
as a model to assess the practical feasibility of the RMD approach
for determining the fetal genotype of a locus on chromosome X. The
current RMD analysis is relevant to at-risk pregnant cases, i.e.,
pregnant women who are heterozygous for mutations on chromosome X
and are carrying male fetuses. Hence, we studied the plasma samples
from ten pregnant women who were heterozygous for the SNP on
chromosome X and were carrying male fetuses. We developed an
allele-discriminative digital real-time PCR assay to measure the
concentrations of the A- and T-allele in each sample. We further
measured the fractional fetal DNA concentrations with the ZFY/X
assay. The digital RMD result is shown in table 1100 of FIG. 11,
which shows fetal genotyping for rs6528633 in maternal plasma by
digital RMD.
[0143] For all of the cases, the fetal SNP genotypes were
concordant with the SPRT classification. The fractional fetal DNA
concentrations (fetal % in table 1100) ranged from 5% to 24%. The
result hence confirmed the feasibility of the digital RMD
strategy.
[0144] Digital RMD for Hemophilia Mutation Detection in DNA
Mixtures
[0145] We next applied the digital RMD approach for hemophilia
mutation detection. We developed seven duplex digital real-time PCR
assays to detect three mutations in the F8 gene, four mutations in
the F9 gene and their corresponding wild-type counterparts. We
evaluated the performance of the digital PCR assays by constructing
artificial DNA mixtures that simulated the composition of maternal
plasma samples with a minority male fetal DNA component amongst a
majority maternal DNA background. We mixed 10% or 20% of placental
DNA obtained from an unaffected male fetus with blood cell DNA
obtained from women heterozygous for the corresponding mutations.
FIG. 12 shows the validation of digital RMD assays with artificial
DNA mixtures. The artificial mixtures were constructed to simulate
the fetal and maternal DNA compositions in maternal plasma. As
shown in table 1200 of FIG. 12, the genotypes of the placental DNA,
which mimicked the fetal DNA in maternal plasma, were correctly
detected in all of the DNA mixtures by digital RMD analysis.
[0146] Detection of Fetal Hemophilia Mutations in Maternal
Plasma
[0147] We tested the digital RMD method for detecting fetal
genotypes for the hemophilia mutations through maternal plasma DNA
analysis. We carried out digital PCR on 12 plasma samples obtained
from seven pregnant women heterozygous for the causative mutations
(TABLE 900). All of the cases involved male fetuses. We also
measured the fractional fetal DNA concentrations in the maternal
plasma samples by the ZFY/X assay. The digital RMD results are
shown in table 1300 of FIG. 13, which shows non-invasive detection
of fetal hemophilia mutations in maternal plasma by digital
RMD.
[0148] The fetal genotypes were correctly classified in all studied
cases by the SPRT algorithm (FIG. 14). For three of the cases
(H26a, H25a and H12a), the fetal DNA proportions were less than
10%. Hence, the degree of quantitative difference between the
amount of mutant and the wild-type alleles was too small to be
classified with data from one 4590-well digital PCR set. Additional
4590-well digital PCR sets were therefore performed until
classifications could be made.
[0149] As controls, we also studied five maternal plasma samples
obtained from normal pregnant women using each of the
mutation-specific assays. FIG. 15 shows digital RMD result for
maternal plasma samples from normal pregnancies. As shown in table
1500 of FIG. 15, no mutant alleles were detected in most of the
cases. For six of the 35 studied maternal plasma cases, the
positive wells containing the mutant alleles constituted less than
0.3% of the total number of positive wells in the experiments.
These positive signals might have resulted from cross
hybridizations of the fluorescent probes during PCR. Nonetheless,
such low numbers of mutant-positive wells would not skew the
allelic ratio between mutant and wild-type alleles to an extent
that would alter the RMD classification by SPRT.
Discussion
[0150] In this study, we have developed noninvasive prenatal
diagnostic strategies to directly detect causative mutations
carried by male fetuses in pregnancies at-risk of X-linked
diseases, using hemophilia as an example. By using the digital RMD
approach for genetic loci on chromosome X, we have accurately
identified the mutant or the wild-type alleles inherited by the
male fetuses in all of the 12 studied maternal plasma samples from
seven pregnant carriers of hemophilia (table 1300). The fetal
genotypes could be detected as early as the 11.sup.th week of
gestation (table 900), demonstrating the potential for early
diagnostic use of the method. The approach using a target region
and a reference region on chromosome X can also be used.
[0151] This noninvasive prenatal mutation detection method could be
combined with the existing noninvasive fetal sex determination test
to further minimize the number of at-risk pregnant cases that would
require invasive diagnostic testing. The identification of affected
fetuses could also facilitate subsequent obstetric management for
pregnant women who would not otherwise consider invasive prenatal
testing. Three to four percent of infants with hemophilia
experience a cranial bleed (Kulkarni R, Lusher J M., J Pediatr
Hematol Oncol., 21:289-295 (1999)) that occurs during labor and
delivery. Prolonged labor and difficult instrumental deliveries are
the main risk factors for this complication (Kadir R A et al.,
Haemophilia., 6:33-40 (2000); Chi C et al., Haemophilia., 14:56-64
(2008)) and should be avoided for delivery of affected fetuses (Lee
C A, Chi C, Pavord S R, et al., Haemophilia., 12:301-336 (2006)).
It is also recommended that affected fetuses are delivered in a
tertiary unit with an affiliated hemophilia center to ensure
availability of necessary expertise and resources for their
management (Lee C A, Chi C, Pavord S R, et al., Haemophilia.,
12:301-336 (2006)). Recently, prenatal diagnosis by third trimester
amniocentesis has been suggested to help appropriate planning of
the mode and place of delivery for parents who are unwilling to
accept the risk of fetal loss associated with earlier prenatal
testing (Chi C, Kadir R A., Obstetric Management. In: Lee C A,
Kadir R A, Kouides P A, eds. Inherited Bleeding Disorders in Women,
Chichester, West Sussex, UK: Wiley-Blackwell, 122-148 (2009)). If a
fetus is unaffected, labor and delivery can be managed without any
restrictions in local maternity units. However, third trimester
amniocentesis is also an invasive procedure and associated with
potential risks and complications (Hodor J G, Poggi S H, Spong C Y,
et al., Am J Perinatol., 23:177-180 (2006); O'Donoghue K et al.,
Prenat Diagn., 27:1000-1004 (2007)). Fetal DNA concentration is the
highest during the third trimester of pregnancy (Lun F M F et al.,
Clin Chem., 54:1664-1672 (2008)), thus embodiments can offer an
accurate noninvasive alternative to third trimester amniocentesis
for this purpose.
VII. COMPUTER SYSTEM
[0152] Any of the computer systems mentioned herein may utilize any
suitable number of subsystems. Examples of such subsystems are
shown in FIG. 16 in computer apparatus 1600. In some embodiments, a
computer system includes a single computer apparatus, where the
subsystems can be the components of the computer apparatus. In
other embodiments, a computer system can include multiple computer
apparatuses, each being a subsystem, with internal components.
[0153] The subsystems shown in FIG. 16 are interconnected via a
system bus 1675. Additional subsystems such as a printer 1674,
keyboard 1678, fixed disk 1679, monitor 1676, which is coupled to
display adapter 1682, and others are shown. Peripherals and
input/output (I/O) devices, which couple to I/O controller 1671,
can be connected to the computer system by any number of means
known in the art, such as serial port 1677. For example, serial
port 1677 or external interface 1681 can be used to connect
computer system 1600 to a wide area network such as the Internet, a
mouse input device, or a scanner. The interconnection via system
bus 1675 allows the central processor 1673 to communicate with each
subsystem and to control the execution of instructions from system
memory 1672 or the fixed disk 1679, as well as the exchange of
information between subsystems. The system memory 1672 and/or the
fixed disk 1679 may embody a computer readable medium. Any of the
values mentioned herein can be output from one component to another
component and can be output to the user.
[0154] A computer system can include a plurality of the same
components or subsystems, e.g., connected together by external
interface 1681 or by an internal interface. In some embodiments,
computer systems, subsystem, or apparatuses can communicate over a
network. In such instances, one computer can be considered a client
and another computer a server, where each can be part of a same
computer system. A client and a server can each include multiple
systems, subsystems, or components.
[0155] It should be understood that any of the embodiments of the
present invention can be implemented in the form of control logic
using hardware and/or using computer software in a modular or
integrated manner. Based on the disclosure and teachings provided
herein, a person of ordinary skill in the art will know and
appreciate other ways and/or methods to implement embodiments of
the present invention using hardware and a combination of hardware
and software.
[0156] Any of the software components or functions described in
this application may be implemented as software code to be executed
by a processor using any suitable computer language such as, for
example, Java, C++ or Perl using, for example, conventional or
object-oriented techniques. The software code may be stored as a
series of instructions or commands on a computer readable medium
for storage and/or transmission, suitable media include random
access memory (RAM), a read only memory (ROM), a magnetic medium
such as a hard-drive or a floppy disk, or an optical medium such as
a compact disk (CD) or DVD (digital versatile disk), flash memory,
and the like. The computer readable medium may be any combination
of such storage or transmission devices.
[0157] Such programs may also be encoded and transmitted using
carrier signals adapted for transmission via wired, optical, and/or
wireless networks conforming to a variety of protocols, including
the Internet. As such, a computer readable medium according to an
embodiment of the present invention may be created using a data
signal encoded with such programs. Computer readable media encoded
with the program code may be packaged with a compatible device or
provided separately from other devices (e.g., via Internet
download). Any such computer readable medium may reside on or
within a single computer program product (e.g. a hard drive, a CD,
or an entire computer system), and may be present on or within
different computer program products within a system or network. A
computer system may include a monitor, printer, or other suitable
display for providing any of the results mentioned herein to a
user.
[0158] Any of the methods described herein may be totally or
partially performed with a computer system including a processor,
which can be configured to perform the steps. Thus, embodiments can
be directed to computer systems configured to perform the steps of
any of the methods described herein, potentially with different
components performing a respective steps or a respective group of
steps. Although presented as numbered steps, steps of methods
herein can be performed at a same time or in a different order.
Additionally, portions of these steps may be used with portions of
other steps from other methods. Also, all or portions of a step may
be optional. Additionally, any of the steps of any of the methods
can be performed with modules, circuits, or other means for
performing these steps.
[0159] The specific details of particular embodiments may be
combined in any suitable manner without departing from the spirit
and scope of embodiments of the invention. However, other
embodiments of the invention may be directed to specific
embodiments relating to each individual aspect, or specific
combinations of these individual aspects.
[0160] The above description of exemplary embodiments of the
invention has been presented for the purposes of illustration and
description. It is not intended to be exhaustive or to limit the
invention to the precise form described, and many modifications and
variations are possible in light of the teaching above. The
embodiments were chosen and described in order to best explain the
principles of the invention and its practical applications to
thereby enable others skilled in the art to best utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated.
[0161] A recitation of "a", "an" or "the" is intended to mean "one
or more" unless specifically indicated to the contrary.
[0162] All patents, patent applications, publications, and
descriptions mentioned above are herein incorporated by reference
in their entirety for all purposes. None is admitted to be prior
art.
Sequence CWU 1
1
36125DNAArtificial Sequencesynthetic locus ZFY/X direct PCR
oligonucleotide F-primer 1caagtgctgg actcagatgt aactg
25228DNAArtificial Sequencesynthetic locus ZFY/X direct PCR
oligonucleotide R-primer 2tgaagtaatg tcagaagcta aaacatca
28316DNAArtificial Sequencesynthetic locus ZFY/X
X-probemodified_base(1)..(1)t modified by fluorescent reporter dye
VICmodified_base(16)..(16)a modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 3tctttagcac attgca
16417DNAArtificial Sequencesynthetic locus ZFY/X
Y-probemodified_base(1)..(1)t modified by luorescent reporter dye
FAMmodified_base(17)..(17)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 4tctttaccac actgcac
17523DNAArtificial Sequencesynthetic locus SNP rs6528633 direct PCR
oligonucleotide F-primer 5ggaagaccaa aaagggataa agg
23621DNAArtificial Sequencesynthetic locus SNP rs6528633 direct PCR
oligonucleotide R-primer 6caccctactc ccagccaatt t
21720DNAArtificial Sequencesynthetic locus SNP rs6528633
T-probemodified_base(1)..(1)t modified by fluorescent reporter dye
VICmodified_base(20)..(20)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 7tgagatatga tatggtcatg
20820DNAArtificial Sequencesynthetic locus SNP rs6528633
A-probemodified_base(1)..(1)t modified by fluorescent reporter dye
FAMmodified_base(20)..(20)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 8tgagatatga taaggtcatg
20920DNAArtificial Sequencesynthetic locus F8 c.826G>A direct
PCR oligonucleotide F-primer 9tggatgccac aggaaatcag
201020DNAArtificial Sequencesynthetic locus F8 c.826G>A direct
PCR oligonucleotide R-primer 10cttcaggagt ggtgcccatt
201117DNAArtificial Sequencesynthetic locus F8 c.826G>A
G-probemodified_base(1)..(1)c modified by fluorescent reporter dye
VICmodified_base(17)..(17)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 11ctattggcat gtgattg
171217DNAArtificial Sequencesynthetic locus F8 c.826G>A
A-probemodified_base(1)..(1)c modified by fluorescent reporter dye
FAMmodified_base(17)..(17)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 12ctattggcat atgattg
171322DNAArtificial Sequencesynthetic locus F8 c.1171C>T direct
PCR oligonucleotide F-primer 13tggatgtggt caggtttgat ga
221423DNAArtificial Sequencesynthetic locus F8 c.1171C>T direct
PCR oligonucleotide R-primer 14ttttaggatg cttcttggca act
231517DNAArtificial Sequencesynthetic locus F8 c.1171C>T
C-probemodified_base(1)..(1)c modifies by fluorescent reporter dye
FAMmodified_base(17)..(17)a modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 15ctgagcgaat ttggata
171616DNAArtificial Sequencesynthetic locus F8 c.1171C>T
T-probemodified_base(1)..(1)c modified by fluorescent reporter dye
VICmodified_base(16)..(16)t modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 16ctgagcaaat ttggat
161724DNAArtificial Sequencesynthetic locus F8 c.6278A>G direct
PCR oligonucleotide F-primer 17tttcaggagg tagcacatac attt
241820DNAArtificial Sequencesynthetic locus F8 c.6278A>G direct
PCR oligonucleotide R-primer 18tgccgtgaat aatcattggt
201916DNAArtificial Sequencesynthetic locus F8 c.6278A>G
A-probemodified_base(1)..(1)c modified by fluorescent reporter dye
VICmodified_base(16)..(16)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 19caacagatcc acctac
162015DNAArtificial Sequencesynthetic locus F8 c.6278A>G
G-probemodified_base(1)..(1)a modified by fluorescent reporter dye
FAMmodified_base(15)..(15)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 20aacagaccca cctac
152124DNAArtificial Sequencesynthetic locus F9 c.802T>A direct
PCR oligonucleotide F-primer 21tctgtggagg ctctatcgtt aatg
242226DNAArtificial Sequencesynthetic locus F9 c.802T>A direct
PCR oligonucleotide R-primer 22acctgcgaca actgtaattt taacac
262315DNAArtificial Sequencesynthetic locus F9 c.802T>A
T-probemodified_base(1)..(1)t modified by fluorescent reporter dye
VICmodified_base(15)..(15)a modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 23tgcccactgt gttga
152415DNAArtificial Sequencesynthetic locus F9 c.802T>A
A-probemodified_base(1)..(1)c modified by fluorescent reporter dye
FAMmodified_base(15)..(15)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 24ctgcccacag tgttg
152524DNAArtificial Sequencesynthetic locus F9 c.874delC direct PCR
oligonucleotide F-primer 25tgtcgcaggt gaacataata ttga
242625DNAArtificial Sequencesynthetic locus F9 c.874delC direct PCR
oligonucleotide R-primer 26ggtgaggaat aattcgaatc acatt
252716DNAArtificial Sequencesynthetic locus F9 c.874delC
C-probemodified_base(1)..(1)a odified by fluorescent reporter dye
VICmodified_base(16)..(16)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 27acatacagag caaaag
162815DNAArtificial Sequencesynthetic locus F9 c.874delC
del-probemodified_base(1)..(1)c modified by fluorescent reporter
dye FAMmodified_base(15)..(15)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 28catacagaga aaagc
152921DNAArtificial Sequencesynthetic locus F9 c.1069G>A direct
PCR oligonucleotide F-primer 29cctcaaattt ggatctggct a
213020DNAArtificial Sequencesynthetic locus F9 c.1069G>A direct
PCR oligonucleotide R-primer 30gctgatctcc ctttgtggaa
203116DNAArtificial Sequencesynthetic locus F9 c.1069G>A
G-probemodified_base(1)..(1)a modified by fluorescent reporter dye
VICmodified_base(16)..(16)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 31actcttcccc agccac
163217DNAArtificial Sequencesynthetic locus F9 c.1069G>A
A-probemodified_base(1)..(1)a modified by fluorescent reporter dye
FAMmodified_base(17)..(17)t modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 32actcttctcc agccact
173327DNAArtificial Sequencesynthetic locus F9 c.1144T>C direct
PCR oligonucleotide F-primer 33cagtacctta gagttccact tgttgac
273429DNAArtificial Sequencesynthetic locus F9 c.1144T>C direct
PCR oligonucleotide R-primer 34catgttgtta tagatggtga actttgtag
293513DNAArtificial Sequencesynthetic locus F9 c.1144T>C
T-probemodified_base(1)..(1)c modified by fluorescent reporter dye
VICmodified_base(13)..(13)g modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 35ccacatgtct tcg
133614DNAArtificial Sequencesynthetic locus F9 c.1144T>C
C-probemodified_base(1)..(1)a modified by fluorescent reporter dye
FAMmodified_base(14)..(14)c modified by minor groove binding
nonfluorescent quencher (MGBNFQ) 36agccacacgt cttc 14
* * * * *