Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Reexamination Certificate
1999-11-29
2002-08-06
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
C436S086000, C436S501000, C436S510000, C436S518000, C436S817000, C436S534000, C436S536000, C436S547000, C436S548000, C436S808000, C436S813000, C436S814000, C436S818000, 43, 43, 43, 43, 43, 43, 43, 43, C424S001110, C424S120000
Reexamination Certificate
active
06429018
ABSTRACT:
TECHNICAL FIELD
This invention relates to a Down's syndrome screening test for pregnant women.
A triple screen of &agr;-fetoprotein, chorionic gonadotropin and unconjugated estrogen in serum has been suggested for the prenatal diagnosis of Down's syndrome (Bennett, J. C., and Plum, F.,
Cecil's Textbook of Medicine
, W. B. Saunders, Philadelphia, 1996, p. 165). However, it allows detection of only 60 to 65% of fetuses with the genetic disorder and gives 5% false positive results. It is also limited to the second trimester of pregnancy (15 to 24 weeks of gestation), and has become expensive as significant license fees are being levied on laboratories running human chorionic gonadotropin analyses using conventional methods (Auxter, S.,
Clin. Labor News
23: 1-3 (1997)).
Definitive prenatal diagnosis of fetal chromosome abnormalities leading to Down's syndrome, which affect 1 in 700 live births, typically involves instead culture of amniocytes at midtrimester gestation. The procedure involves the aspiration of a small sample of amniotic fluid (amniocentesis), culturing of the fetal cells contained in the fluid, and determination of the karyotype of these cells and thus the fetus. The major indications for the use of this technique for the detection of chromosome abnormalities are maternal age (usually offered to all mothers over the age of 35 at the time of expected delivery), the presence of a parental chromosome abnormality, or a maternal history of carrying a previous trisomic child or aborted fetus karyotyped to be trisomic. Direct transcervical aspiration of chorionic villi (chorionic villus sampling, or CVS) has also been used for prenatal diagnosis.
Though both procedures have been shown to be relatively safe and reliable, it is generally accepted that they involve some risk, including risk of miscarriage, and, in the case of CVS, also risk of limb hypoplasia in babies born following the procedure. It would be desirable to have other methods for screening pregnant women for Down's syndrome fetuses, particularly screens that are noninvasive and sensitive. Most Down's syndrome cases occur in younger pregnant women, those under 35 at the time of expected delivery, or the majority of pregnancies. Less invasive screening tests are needed employing serum or urine samples to identify those at high risk for Down's syndrome pregnancies, who may not want the risk of amniocentesis or CVS.
BACKGROUND OF THE INVENTION
Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted in relatively large quantities by the trophoblast cells of the placenta (Masure, H. R., et al.,
J. Clin. Endocrinol. Metab
. 53: 1014-1020 (1981)). hCG is composed of two dissimilar subunits, &agr; (92 amino acids and two N-linked oligosaccharides) and &bgr; (145 amino acids and two N-linked and four O-linked oligosaccharides), joined noncovalently, and is detected in the serum and urine of pregnant women and in those with trophoblast disease (hydatidiform mole and choriocarcimoma). Free &agr;- and free &bgr;-subunits, and degraded hCG and free &bgr;-subunit molecules are also detected in serum and urine samples (Birken, S., et al.,
Endocrinology
122: 572-583 (1988)). The degraded molecules include nicked hCG and nicked free &bgr;-subunit, each cleaved between &bgr;-subunit residues 47 and 48 (or less commonly between residues 43 and 44 or 44 and 45), nicked &bgr;-subunit missing all or part of the C-terminal sequence (&bgr;93-145) and, a terminal degradation product comprising two fragments, &bgr;-subunit sequences 6-40 and 55-92, held together by disulfide linkages, found primarily in urine samples (FIG.
1
). The terminal degradation product has no O-linked oligosaccharides and degraded N-linked oligosaccharide moieties, one or two N-linked pentasaccharides, versus two undecasaccharides on free &bgr;-subunit and hCG (FIG.
2
A). The terminal degradation product was called &bgr;-core fragment (&bgr;-core, Blithe, D., et al.,
Endocrinology
122: 173-180 (1988)), firstly because of its small size (~9,000 daltons; hCG is 37,000 daltons), and secondly because of its retention of hCGB radioimmunoassay or &bgr;-submit core antisera (versus C-terminal or other) immunoreactivity (Birken, et al., and Masure, et al., cited above). Through most of a pregnancy, &bgr;-core fragment is the principal hCG &bgr;-subunit-related molecule in urine samples.
Serum and urine free &bgr;-subunit derive from three sources: direct section by trophoblast cells, slow dissociation of circulating hCG into free &agr;- and &bgr;-subunits, and by the nicking of hCG by macrophage or neutrophil enzymes associated with trophoblast tissue, and the more rapid dissociation of nicked hCG in circulation (FIG.
1
). Free &bgr;-subunit may be nicked by nicking enzymes in the circulation. Urine &bgr;-core fragment appears to derive from the degradation of nicked free &bgr;-subunit in the kidney.
In the late 1980s, the triple marker test mentioned above was developed to screen for Down syndrome pregnancies. It combined maternal age with serum measurements of hCG, &agr;-fetoprotein, and unconjugated estriol (Bogart, M. H., et al.,
Prenat. Diagn
. 7:623-630 (1987), U.S. Pat. No. 4,874,693 to Bogart, Wald, N. J., et al.,
Br. J. Obstet. Gynaecol
. 95: 334-341 (1988), and Canick, J. A.,
J. Clin. Immunoassay
13: 30-33 (1990)). More recently, serum-free &bgr;-subunit tests and free &bgr;-subunit-&agr;-fetoprotein combinations have been introduced as alternative Down syndrome-screening methods (Macri, J. N., el al.,
Am. J. Obstet. Gynecol
. 163: 1248-1253 (1990) and Spencer, K., et al.,
Ann. Clin. Biochem
. 30: 394-401 (1993)). The best serum free &bgr;-subunit combination, or the optimal triple marker test, however, detects only 60 to 65 percent of Down's syndrome cases, with a 5 percent false-positive rate. At these detection and false-positive rates, approximately 80 amniocenteses need to be performed to detect a single case of Down syndrome, and a significant number of Down's syndrome cases are missed (Cole, L. A., et al.,
Prenatal Diagnosis
17: 607-614 (1997)). There is a need for improvement in prenatal screening methods.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a prenatal screening test for Down's syndrome pregnancies.
It is another and more specific object of the invention to provide a sensitive, noninvasive test for Down's syndrome fetuses in pregnant women.
It is a further object of the invention to provide an improvement in the triple marker test that exhibits a decreased false positive rate.
These and other objects are accomplished by the present invention, which provides a novel diagnostic method for screening for the presence or absence of Down's syndrome in the fetus of a pregnant woman which comprises obtaining a biological test sample from the woman and determining the presence of Down's syndrome by observation of hyperglycosylated gonadotropin in the sample. This typically involves measuring the concentration of hyperglycosylated gonadotropin, its free &bgr;-subunit, its free &agr;-subunit, and/or its &bgr;-core fragment in the test sample, and determining the presence of Down's syndrome by observation that the concentration in the test sample population differs from a normal hyperglycosylated gonadotropin or free &agr;-subunit, free &bgr;-subunit, or &bgr;-core fragment population and/or is the same as, or similar to, a Down's syndrome population. In preferred embodiments, the test sample is urine, saliva, plasma or serum, the population comprises hyperglycosylated hCG, &bgr;-core fragment, free &agr;-subunit, free &bgr;-subunit, and mixtures thereof, and any differences between the properties observed in the normal and Down's syndrome samples reflect differences observed in the carbohydrate content of the glycopolypeptides and/or glycopeptides.
Carbohydrate compositional or structural analyses, immunoassays, and combinations of these methods are generally employed. In some embodiments, hyperglycosylated gonadotrop
Cole Laurence A.
Kardana Andrew
Daniels-Cook Lisa V.
Krinsky Mary M.
Le Long V.
Yale University
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