Riverbend DS Assocation Home Page » Resources » Patents » Prenatal Screening » Prenatal Down Syndrome Screening with Assays Specific for UGP Prenatal Down Syndrome Screening with Assays Specific for UGP |
Inventors: Cuckle; Howard S. (North Yorkshire, GB); Walker; Roger P. (Benicia, CA) Assignee: Chiron Diagnostics Corporation (Medfield, MA) Appl. No.: 647437 Filed: April 30, 1996 |
Primary Examiner: Woodward; Michael P. Assistant Examiner: Brumback; Brenda Glass Attorney, Agent or Firm: Lauder; Leona L., Morgenstern; Arthur S. United States Patent 5,716,853 February 10, 1998 |
Claims
What we claim is:
1. A method for prenatally determining whether
there is a significant risk of a pregnancy being affected by Down syndrome
comprising:
(a) taking a maternal urine sample during the first
trimester of said pregnancy;
(b) testing said maternal urine sample to
determine the level of UGP in said sample with an immunoassay that is specific
for UGP and has a molar cross-reactivity of less than about 10% with each of the
following: intact hCG, .beta.-subunit hCG, and .alpha.-subunit hCG;
(c)
determining whether the level of UGP in said sample is elevated above a level of
UGP that is normal in urine samples from women whose pregnancies are unaffected
by aneuploidy, and whose pregnancies are at about the same gestational age as
the pregnancy under analysis; and
(d) determining that there is a
significant risk of fetal Down syndrome if the level of UGP in said sample is
elevated above said normal level.
2. The method according to claim 1
wherein said immunoassay has a molar cross-reactivity of less than about 5% with
each of the following: intact hCG, .beta.-subunit of hCG, and .alpha.-subunit
hCG.
3. The method according to claim 1 wherein said immunoassay has a
molar cross-reactivity of less than about 3% with each of the following: intact
hCG, .beta.-subunit hCG, and .alpha.-subunit hCG.
4. The method
according to claim 1 wherein said immunoassay has a molar cross-reactivity of
less than about 1% with each of the following: intact hCG, .beta.-subunit hCG,
and .alpha.-subunit hCG.
5. The method according to claim 1 wherein said
maternal urine sample was taken during the fifth week to the 14th completed week
of said pregnancy.
6. The method according to claim 5 wherein said
maternal urine sample was taken during the fifth week to the 13th completed week
of said pregnancy.
7. The method according to claim 5 wherein said
maternal urine sample was taken during the sixth week to the 14th completed week
of said pregnancy.
8. The method according to claim 5 wherein said
maternal urine sample was taken during the seventh week to the 14th completed
week of said pregnancy.
9. The method according to claim 5 wherein said
maternal urine sample was taken during the eighth week to the 14th completed
week of said pregnancy.
10. The method according to claim 5 wherein said
maternal urine sample was taken during the ninth week to the 14th completed week
of said pregnancy.
11. The method according to claim 5 wherein said
maternal urine sample was taken during the 10th week to the 14th completed week
of said pregnancy.
12. The method according to claim 5 wherein said
maternal urine sample was taken during the ninth week to the thirteenth
completed week of said pregnancy.
13. The method according to claim 5
wherein said maternal urine sample was taken during the ninth week to the
twelfth completed week of said pregnancy.
14. The method according to
claim 1 wherein said immunoassay is in a sandwich assay format or in a
competitive assay format.
15. The method according to claim 1 wherein
said immunoassay is automated.
16. The method according to claim 1
wherein the level of UGP in said sample is elevated above said normal level to a
statistically significant extent.
17. The method according to claim 1
wherein the level of UGP in said sample is two or more times the level of UGP
that is normal for pregnancies unaffected by aneuploidy at about the same
gestational age as the pregnancy sample under analysis.
18. The method
according to claim 1 wherein the UGP levels are corrected for variability in
urine concentrations by being expressed relative to creatinine content in said
urine samples.
19. A method according to claim 1 wherein said
immunoassay comprises the use of monoclonal antibodies and/or affinity-purified
polyclonal antibodies that bind specifically to UGP.
20. A method
according to claim 1 wherein said immunoassay comprises the use of antibodies
directly or indirectly linked to a detectable marker, and wherein signal from
said marker indicates the level of UGP, and said signal's intensity is directly
proportional to the level of UGP in said sample.
21. The method
according to claim 20 wherein said immunoassay further comprises the use of
antibodies linked to a solid phase.
22. The method according to claim 20
wherein said detectable marker is selected from the group consisting of
radionuclides, fluorescers, bioluminescers, chemiluminescers, dyes, enzymes,
coenzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme
subunits, metal ions, and free radicals.
23. The method according to
claim 22 wherein the detectable marker is either selected from the group
consisting of acridinium esters, acridinium sulfonyl carboxamides, fluorescein,
luminol, umbelliferone, isoluminol derivatives, photoproteins, and luciferases,
or is produced by an enzymatic reaction upon a substrate.
24. The method
according to claim 20 wherein the detectable marker is either an acridinium
ester or is produced by an enzymatic reaction with a chemiluminescent substrate
and an enzyme selected from the group consisting of alkaline phosphatase,
glucose oxidase, glucose 6-phosphate dehydrogenase, .alpha.,
.beta.-galactosidase, horseradish peroxidase, and xanthine oxidase.
25.
The method according to claim 21 wherein said solid phase comprises magnetic or
paramagnetic particles.
26. The method according to claim 25 wherein
said immunoassay is automated, and wherein said detectable marker is an
acridinium ester.
Description
This application claims the benefit of U.S. Provisional Application No.
60/000,945 filed Jul. 7, 1995 which is entitled "Urinary Screening for Down's
Syndrome and Other Aneuploidies."
FIELD OF THE INVENTION
The
present invention is in the field of prenatal diagnosis. It concerns
non-invasive methods to screen prenatally for fetal Down syndrome by
immunoassays employing antibodies that are specific for urinary gonadotropin
peptide (UGP) also known as .beta.-core or urinary gonadotropin fragment
(UGF).
BACKGROUND OF THE INVENTION
Trisomy 21, commonly known
as Down syndrome, is characterized by an extra copy of chromosome 21. People
afflicted with Down syndrome have severe mental retardation, reduced life
expectancies, and abnormal immune responses that predispose them to serious
infections as well as thyroid autoimmunity. Further, 40% of Down syndrome
patients have congenital heart disease and a 10 to 20-fold increased risk of
developing leukemia over the general population. All Down syndrome patients
older than 40 develop neuropathological changes characteristic of Alzheimer's
disease.
Prenatal tests to detect aneuploidy, such as trisomy 21, by
amniocentesis or chorionic villus sampling (CVS) have been available since the
late 1960s. Amniocentesis is the most common invasive prenatal diagnostic
procedure. In amniocentesis, amniotic fluid is sampled by inserting a hollow
needle through the mother's anterior abdominal and uterine walls into the
amniotic cavity by piercing the chorion and amnion. It is usually performed in
the second trimester of pregnancy. CVS is performed primarily during the first
trimester, and involves collecting cells from the chorion which develops into
the placenta.
Another invasive prenatal diagnostic technique is
cordocentesis or percutaneous umbilical cord blood sampling, commonly known as
fetal blood sampling. Fetal blood sampling involves obtaining fetal blood cells
from vessels of the umbilical cord, and is often performed about the 20th
gestational week.
Amniocentesis is used selectively because it presents
a risk of about 1% of inducing spontaneous abortion. CVS and fetal blood
sampling carry a similar or higher risk of inducing abortion, and there is also
concern that these procedures may lead to fetal limb malformations in some
cases. Thus, amniocentesis, CVS and fetal blood sampling are procedures that are
only employed if a pregnancy is considered at high risk for a serious congenital
anomaly. Thus, some means is required to select those pregnancies that are at a
significant risk of Down syndrome to justify the risks of such invasive prenatal
diagnostic procedures, as amniocentesis, CVS and fetal blood sampling.
Prior to 1983, the principal method for selecting pregnancies that had
an increased risk for Down syndrome was based on maternal age, that is, the
older the age of the mother, the higher the risk that the pregnancy would be
affected by Down syndrome. In 1974, biochemical screening for neural tube
defects by measuring alpha-fetoprotein (AFP) began. In 1984, the use of the AFP
screen was additionally adopted for the detection of Down syndrome. Since the
early 1990s, a multiple marker blood test has been used to screen for this
disorder. A common version of this test is the three marker triple test. The
triple screen measures AFP, human chorionic gonadotropin (hCG) and unconjugated
estriol (uE.sub.3) in the serum of pregnant women.
The triple screen
provides a means to screen the population of pregnant women to determine which
pregnancies are at risk for Down syndrome and other serious genetic defects. The
risk is calculated based on the results of the screen, along with other
cofactors, such as, maternal age, to determine if the risk is high enough to
warrant an invasive diagnostic procedure, such as, amniocentesis, CVS or fetal
blood sampling. Such prenatal screens, as the triple screen, can be used either
to reduce the need for amniocentesis or to increase Down syndrome detection for
the same amount of amniocentesis. "The efficiency of the Triple test is
projected to be one case of fetal Down syndrome detected for every 50
amniocenteses performed." Canick and Knight, "Multiple-marker Screening for
Fetal Down Syndrome," Contemporary OB/GYN, pp. 3-12 (April 1992).
Although pregnant women who are 35 years or older are the standard high
risk group for fetal Down syndrome, screening also needs to be applied to young
women because although they are at lower risk, most affected pregnancies are in
young women. Approximately 80% of babies born with Down syndrome are born to
mothers under 35. "Down Syndrome Screening Suggested for Pregnant Women under
35, "ACOG Newsletter, 38(8): 141 (August 1994).
The triple screen
combines the analysis of three markers from serum to reduce false positive
results (which result in the performance of unnecessary invasive procedures) and
false negatives (in which serious genetic defects, such as, trisomy 21, go
undetected). In women under 35, the double screen (AFP and hCG) can pick up
about half of Down syndrome cases and a large proportion of other chromosome
defects during the second trimester. The triple screen (AFP, hCG and uE.sub.3)
increases the detection rate by 5-10% of Down syndrome and a further increase in
the detection of all other serious chromosome defects, thus decreasing the
number of false-positives. Such rates mean that the double and triple screens
still fail to detect a significant number of Down syndrome affected pregnancies.
Although the triple screen has a suggested screening period of 15 to 20
weeks gestation, such screening has been recommended between weeks 16-18 to
maximize the window for spinal bifida detection. Canick and Knight, supra
(April 1992). A 1992 survey of prenatal maternal serum screening for AFP alone
or for multiple analyses reported that very few such screenings occurred in the
thirteenth or earlier week of gestation. Palomaki et al., "Maternal Serum
Screening for Fetal Down Syndrome in the United States: A 1992 Survey," Am. J.
Obstet. Gynecol., 169(6): 1558-1562 (1992). The triple screen thus suffers from
the additional problem that once a risk of Down syndrome is predicted, and
amniocentesis or another invasive prenatal definitive diagnostic procedure is
performed to diagnose Down syndrome, it is at an advanced date of gestation,
when termination of a pregnancy can be more physically and emotionally trying
for the mother, and when certain less traumatic abortion procedures, such as,
vacuum curettage, may not be available.
The limitations of the triple
screen and the adverse consequences of unnecessary, potentially harmful and
expensive invasive prenatal diagnostic procedures, such as, amniocentesis, have
led to a search for more discriminatory markers for prenatal Down syndrome
screening. Cuckle et al., "Urinary D-Core Human Chorionic Gonadotropin: A New
Approach to Down's Syndrome Screening," Prenatal Diagnosis, 14: 953-958 (October
1994), pointed out on page 953 that "human chorionic gonadotropin (hCG) is the
most discriminatory maternal serum marker of Down's syndrome. . . . and
carried out a study to establish whether urinary .beta.-core-hCG an alternative
name for UGP, a major metabolic product of hCG, might be even a better marker."
Human chorionic gonadotropin (hCG) is a glycopeptide hormone produced by
the syncytiotrophoblasts of the fetal placenta that is thought to maintain the
function of the corpus luteum during the first few weeks of pregnancy, to
promote steroidogenesis in the fetoplacental unit, and to stimulate fetal
testicular secretion of testosterone. It can be detected by immunoassay in the
maternal urine within days after fertilization and thus provides the basis of
the most commonly used pregnancy tests. The intact hCG molecule is a dimer
comprising a specific .beta. subunit covalently bound to an e subunit, which is
common to other glycoproteins.
Urinary gonadotropin peptide (UGP) has an
amino acid sequence related to the .beta.-subunit of hCG. UGP is comprised of
.beta.-subunit residues 6 through 40 attached by disulfide linkages to residues
55 through 92. UGP is glycosylated but lacks the sialic acid and O-linked
carbohydrate residues present on hCG .beta. subunit.
UGP has been found
in the urine of pregnant women carrying normal fetuses, and also in the urine of
patients with gestational trophoblastic and non-trophoblastic malignancies Cole
et al., "Urinary Human Chorionic Gonadotropin Free B-subunit and B-core
Fragment: A New Marker of Gynecological Cancers," Cancer Res., 48: 1356-1360
(1988); Cole et al., "Urinary Gonadotropin Fragments (UGF) in Cancers of the
Female Reproductive System," Gynecol. Oncol., 31: 82-90 (1988); O'Connor et al.,
"Development of Highly Sensitive Immunoassays to Measure Human Chorionic
Gonadotropin, Its .beta.-subunit, and .beta.-core Fragment in the Urine:
Application to Malignancies," Cancer Res., 48: 1361-1366 (1988); and Akar et al,
"A Radioimmunoassay for the Core Fragment of the Human Chorionic Gonadotropin
.beta.-subunit," J. Clin. Endocrinol. and Metab., 66: 538-545 (1988). UGP has
also been found to be associated with certain ovarian cancers. Cole and Nam,
"Urinary Gonadotropin Fragment (UGF) Measurements in the Diagnosis and
Management of Ovarian Cancer," Yale J. Bio. and Med., 62: 367-378 (1989).
Cuckle et al., supra examined UGP levels in second trimester samples
from singleton pregnancies affected and unaffected by Down syndrome and compared
the levels. A radioimmunoassay was used in the Cuckle et al. study as described
in Lee et al., "The purification and development of a radioimmunoassay for
.beta.-core fragment of human chorionic gonadotrophin in urine: application as a
marker of gynaecological cancer in premenopausal and postmenopausal women," J.
Endocrinol, 130: 481-489 (1991). That "assay has a partial mole per mole
cross-reaction with intact hCG (6.9 per cent) and free .beta.-hCG (18 per cent)
but negligible cross-reactivity with free .alpha. subunit . . . . " Cuckle et
al. at page 954.
Cuckle et al. showed that UGP levels are elevated on
average in the second trimester of pregnancies affected by fetal Down syndrome
and may be reduced in the presence of other serious, but less common
aneuploidies. The observed median level in Down syndrome (6.11 MOM: 95%
confidence interval 3.7 to 10.0) was greater than the corresponding median level
for intact hCG in maternal serum (2.0 MOM; 1.9-2.1) and free .beta.-hCG (2.3
MOM; 2.1-2.5).
There are important advantages to using urinalysis for
prenatal screening for Down syndrome. Urine tests are less expensive than serum
testing, avoid the safety issues and handling risks associated with the
collection and storage of blood samples, as well as the invasiveness and
discomfort of phlebotomy. Urine samples can be easily collected and shipped, if
necessary, where women have limited access to medical testing facilities because
of geography or socio-economic status. UGP is stable to changes in temperature,
pH, and storage time at -20.degree. and 40.degree. C.
However, the
.beta.-core fragment assay described in Cuckle et al., supra had elevated levels
in Down syndrome affected pregnancies only in samples taken between the 19th
week and the 22nd week plus 4 days of gestation. As indicated above, there are
disadvantages to second trimester testing, in that delays in confirming a fetal
Down syndrome diagnosis result in more traumatic abortion procedures being
necessitated. Also, the emotional attachment and expectations of the pregnant
woman and her family for a healthy baby, grow during the pregnancy, making the
abortion decision more difficult later in the gestational term.
The
instant invention provides the benefits of urinalysis and avoids the problems of
second trimester prenatal screening by providing methods to screen first
trimester urine samples for fetal Down syndrome. The prenatal screening methods
of the instant invention are highly specific for UGP and minimally
cross-reactive with intact hCG, with .beta.-subunit hCG and with .alpha.-subunit
hCG.
SUMMARY OF THE INVENTION
The instant invention provides
methods for prenatally determining whether there is a high risk of a pregnancy
being affected by Down syndrome by testing maternal urine samples for elevations
of urinary gonadotropin peptide (UGP) levels above normal. The methods employ
immunoassays that are specific for UGP and have molar cross-reactivities of less
than about 10% with intact hCG, with .beta.-subunit hCG, and with
.alpha.-subunit hCG. Preferably the immunoassay methods of this invention have a
molar cross-reactivity of less than about 5%, more preferably less than about
3%, and still more preferably less than about 1%, with intact hCG, with
.beta.-subunit hCG, and with .alpha.-subunit hCG.
The UGP level in the
maternal urine sample is related to the median for unaffected pregnancies, and
the degree of elevation or reduction indicates the risk of Down syndrome.
Results from the prenatal screening methods of this invention are generally
expressed as multiples of the median value (MOM) for unaffected pregnancies of
the same gestation. Exemplary positive results from the screening methods
according to this invention are those wherein the UGP level is from about 1.1
MOM to higher multiples, from about 1.5 MOM to higher multiples, from about 2
MOM and higher multiples.
Such a screening result is used to assess the
fetal Down syndrome risk either alone or in conjunction with results from other
screening tests with other serum and/or urinary markers, and/or other factors,
such as, maternal age, maternal health, maternal weight among other factors. For
example, maternal age and UGP levels are independent predictors of Down syndrome
risk, as is true for each of the commonly used serum markers. Therefore, after
performing the prenatal screening methods of this invention, the risk of a Down
syndrome affected pregnancy can be calculated by multiplying the age-related
risk by a likelihood ratio derived from the UGP level found in the maternal
urine sample in relation to control samples.
Other urinary markers which
could be preferred for assessing the risk of a Down syndrome affected pregnancy
in conjunction with UGP levels, include pregnancy-associated plasma protein A
(PAPP-A), dimeric inhibin, total estrogen (tE), unconjugated estriol (uE.sub.3),
total estriol (tE.sub.3), AFP and proform of eosinophilic major basic protein
(proMBP), among other urinary marker possibilities.
In general, a
positive result from the screening methods of this invention is an indicator
that a more invasive prenatal diagnostic procedure, such as, amniocentesis, CVS
or fetal blood sampling, should be performed to determine definitively whether
the pregnancy is affected with Down syndrome.
Gestation-specific medians
for UGP can be calculated by weighted non-linear regression from the values for
control urine samples. To account for variations in the concentrations of urine
samples, UGP levels can be expressed in terms of creatinine. Gestational ages of
cases and controls can be determined by ultrasound parameters and by last
menstrual period dating.
The control samples are preferably taken from a
population of pregnant women that are matched as well as practicable to the
population from which the pregnant woman who provided the test sample comes. For
example, population parameters could include race, ethnicity, and geographical
location, among other parameters.
The prenatal screening methods of this
invention are useful to test first trimester maternal urine samples. Thereby the
screening methods of this invention provide prenatal screening results at a
significantly earlier date than had heretofore been available. The benefits of
such earlier screening results are discussed above in the Background.
Generally, maternal urine samples can be taken for testing, according to
this invention, during the fifth week to the 14th completed week, that is, 14
weeks plus six days, of gestation. Preferably, maternal urine samples can be
taken for testing during the sixth week to the 14th completed week of gestation;
more preferably during the seventh week to the 14th completed week of gestation;
still more preferably during the eighth week to the 14th completed week of
gestation; further preferably, during the ninth week to the 14th completed week
of gestation; alternatively, during the 10th week to the 14th completed week of
gestation; further alternatively during the ninth week to the 13th week of
gestation; and still further also preferably from the ninth week to the twelfth
completed week of gestation.
Other preferred gestational periods from
the first trimester, during which maternal urine samples can be taken for
testing according to this invention include the following: the gestational
periods from the beginning of the seventh week to the end of the 14th week; from
the beginning of the eighth week to the end of the 14th week; from the beginning
of the ninth week to the end of the 14th week; from the beginning of the tenth
week to the end of the 14th week; from the beginning of the fifth week to the
end of the 13th week; from the beginning of the fifth week to the end of the
12th week; from the beginning of the sixth week to the end of the 13th week;
from the beginning of the sixth week to the end of the 12th week; from the
beginning of the seventh week to the end of the 13th week; from the beginning of
the seventh week to the end of the 12th week; from the beginning of the eighth
week to the end of the 13th week; from the beginning of the eighth week to the
end of the 12th week; from the beginning of the eighth week to the end of the
11th week; from the beginning of the eighth week to the end of the 10th week;
from the beginning of the ninth week to the end of the 11th week; from the
beginning of the ninth week to the end of the 10th week; from the beginning of
the tenth week to the end of the 13th week; and from the beginning of the tenth
week to the end of the twelfth week.
The immunoassay methods of this
invention employ UGP standards. Blithe et al., U.S. Pat. No. 5,445,968 (issued
Aug. 29, 1995) discloses methods of purifying UGP.
The prenatal
screening methods of this invention can be in any standard immunoassay format,
for example, a competitive radioimmunoassay, a sandwich EIA or sandwich
radioimmunoassay, among other known formats. A sandwich assay is a preferred
format of this invention, and a sandwich EIA is a further preferred embodiment.
The prenatal screening methods of this invention can be automated. A
preferred automated immunoassay system is Ciba Corning Diagnostic Corp.'s
(CCD's) ACS-180.TM. Automated Chemiluminescence System CCD; Medfield, Mass.
(USA).
The prenatal screening methods of this invention employ
antibodies, that are defined herein to include whole antibodies or biologically
active fragments of antibodies. The antibodies used in the immunoassay methods
can be monoclonal and/or polyclonal, preferably monoclonal and/or
affinity-purified polyclonal antibodies. The specificity of the immunoassay
methods is provided by antibodies which specifically bind to UGP; generally such
antibodies are monoclonal antibodies.
Tracer antibodies that can be used
in the immunoassay methods of this invention can be directly or indirectly
linked to a detectable marker. The signal from said marker can indicate the
level of UGP in the sample tested. The signal's intensity may be directly
proportional to the level of UGP in the sample.
Exemplary detectable
markers can be selected from the group consisting of radionuclides, fluorescers,
bioluminescers, chemiluminescers, dyes, enzymes, coenzymes, enzyme substrates,
enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and free
radicals.
Antibodies used in the immunoassay methods may be linked to a
solid phase, for example, the wall of a container or the surface of magnetic or
paramagnetic particles, among other solid phases.
Source: http://www.uspto.gov/patft/ | |
Revised: February 14, 2001. |