Inventors: Pandian; M. R. (27432 Almendra, Mission Viejo, CA 92691) Assignee: The General Hospital Corporation (Boston, MA) Appl. No.: 229136 Filed: January 12, 1999

Primary Examiner: Warden; Jill Assistant Examiner: Cole; Monique T. Attorney, Agent or Firm: Weiss; Steven M. United States 6,127,186 October 3, 2000

Claims
What is claimed is:

1. A method of determining an increased risk of a woman carrying a Downs Syndrome affected fetus, the method comprising the steps of:

a) quantitatively assaying a sample of a biological fluid from a pregnant woman for an amount of leptin in the sample, thereby determining the amount of leptin in the sample; and

b) comparing the amount of leptin in the sample in step a with an amount of leptin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of leptin in the sample in step a with an amount of leptin found in pregnant women carrying a Downs syndrome affected fetus, thereby determining those at an increased risk of carrying a Downs syndrome affected fetus.

2. The method of claim 1 wherein the amount of leptin in the sample as determined in step a is below the median amount of leptin found in a pregnant women carrying a Downs syndrome unaffected fetus.

3. The method of claim 1 wherein the sample from the pregnant women is selected from the group consisting of serum, plasma or urine.

4. The method of claim 1 wherein the sample is taken from a pregnant women in any of the first or the second or the third trimester of pregnancy.

5. The method of claim 1 wherein the quantitative assay of the sample is performed by immunoassay.

6. The method of claim 1 further comprising the step of analyzing at least one additional analyte predictive of an increased risk of a fetus being affected by Downs syndrome said additional analyte selected from the group consisting of hCG, unconjugated estriol, alpha-fetoprotein, inhibin, PAPP-A, progesterone, DHEA-S, and Leukocyte acid phosphatase.

7. The method of claim 1 further comprising the step of analyzing at least one additional factor predictive of an increased risk of a fetus being affected by Downs syndrome.

8. The method of claim 7 wherein the additional factor is an ultrasound result.

9. A method of determining whether there is an increased risk of a woman carrying a fetus which is affected by Downs Syndrome, the method comprising the steps of:

a) quantitatively assaying a sample of a biological fluid from a pregnant woman for an amount of leptin in the sample, thereby determining the amount of leptin in the sample;

b) comparing the amount of leptin in the sample in step a with an amount of leptin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of leptin in the sample in step a with an amount of leptin found in pregnant women carrying a Downs syndrome affected fetus,

c) quantitatively assaying a sample of a biological fluid from a pregnant woman for an amount of prorenin in the sample, thereby determining the amount of prorenin in the sample; and

d) comparing the amount of prorenin in the sample in step a with an amount of prorenin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of prorenin in the sample in step a with an amount of prorenin found in pregnant women carrying a Downs syndrome affected fetus; and

e) correlating the results of step b and step d to determine those at an increased risk of carrying a Downs syndrome affected fetus.

10. The method of claim 9 wherein the sample from the pregnant women is selected from the group consisting of serum, plasma or urine.

11. The method of claim 9 wherein the sample is taken from a pregnant women in any of the first trimester or the second trimester or the third trimester of pregnancy.

12. The method of claim 9 wherein the quantitative assay of the sample is performed by immunoassay.

13. The method of claim 9 further comprising the step of analyzing at least one additional analyte predictive of an increased risk of a fetus being affected by Downs syndrome.

Description
FIELD OF THE INVENTION

The present invention relates to assay methods which allow for the detection, and quantitation, of analytes; such as, leptin and/or prorenin and/or renin, in biological samples, such as blood or urine from a pregnant woman, which are associated with an increased risk that the pregnant woman's fetus has Downs Syndrome.

BACKGROUND OF THE INVENTION

Trisomy 21, commonly known as Downs syndrome, is characterized by an extra copy of chromosome 21. People afflicted with Downs 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 Downs syndrome patients have congenital heart disease and a 10 to 20-fold increased risk of developing leukemia relative to the general population. All Downs 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 performed about the 20.sup.th 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 Downs syndrome to justify the risks associated with invasive prenatal diagnostic procedures, such as amniocentesis, CVS and fetal blood sampling.

Prior to 1983, the principal method for selecting pregnancies that had an increased risk for Downs syndrome was based on material age, that is, the older the age of the mother, the higher the risk that the fetus would be affected by Downs syndrome. In 1974, biochemical screening for neural tube defects by measuring alpha-fetoprotein (AFP) in serum began. In 1984, the use of the AFP screen was additionally adopted for the detection of Downs 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 Downs 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 Downs syndrome detection for the same number of amniocentesis. "The efficiency of the Triple test is projected to be one case of fetal Downs syndrome detected for every 50 amniocenteses performed." Canick and Knight, "Multiple-marker Screening for Fetal Downs 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 Downs 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 Downs syndrome are born to mothers under 35. ["Downs Syndrome Screening Suggested for Pregnant Women under 35, "ACOG Newsletter, 38(8): 141 (August 1994).]

The triple screen combines the analysis of three serum markers 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 detect about half of Downs 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 of Downs syndrome by 5-10% and a further increase in the detection of all other serious chromosome defects, thus decreasing the number of false-negatives. Such rates mean that the double and triple screens still fail to detect a significant number (30%-35%) of Downs syndrome affected pregnancies.

Other screening markers have been found which may offer some predictive value with respect to Downs Syndrome. The present Applicant has added to this repertoire of predictive markers by finding that leptin, prorenin and/or renin are predictive of a pregnancy being affected by Downs Syndrome.

Leptin has heretofore been associated with obesity. Obesity is the result of a disorder in the body energy balance that occurs when energy intake chronically exceeds energy expenditure. This excess in energy intake is stored in the adipocyte. The recently discovered hormone leptin contributes to the regulation of energy balance by informing the brain of the amount of adipose tissue in the body. The brain may then make the appropriate adjustments in either energy intake or expenditure. Leptin is the protein product of the ob gene and in humans is expressed exclusively in adipose tissue. Studies suggest that leptin is a negative regulator of adiposity. However, leptin has only recently been discovered and further investigations into its actions in humans and its role in obesity remain to be determined. Leptin has also heretofore been generally associated with reproductive function.

Renin is an enzyme that belongs to the family of aspartyl proteases, a classification that is based on the properties of having 2 aspartic acid residues at the active site and its susceptibility to inhibition by pepstatin. Renin synthesis was first discovered in the juxtaglomerular cells of the kidney. At present there is evidence that renin synthesis can also occur in other organs such as brain, heart and arterial smooth muscle. Renin circulates in two different forms, prorenin and the active renin form. Prorenin is the enzymatically inactive biosynthetic precursor of renin. In the secretory granules of the juxtaglomerular cell, prorenin is processed to active renin by a thiol protease resembling cathepsin B. An amino terminal prosegment of 42 amino acids is cleaved from the prorenin which allows the exposure of the active site of renin. Active renin converts angiotensinogen (renin substrate) to the biologically inactive decapeptide angiotensin I. Angiotensin I in turn is converted to the octapeptide angiotensin II by means of the angiotensin converting enzyme (ACE). Angiotensin II causes constriction of the small arteries and also promotes sodium and water reabsorption in tubules both directly and indirectly via aldosterone. Aldosterone is a steroid hormone produced by the adrenal gland and its secretion is stimulated by Angiotensin II. Heretofore, the clinical utility of plasma renin is mainly centered around the diagnosis and management of patients with hypertension due to renal artery stenosis or renovascular hypertension. Approximately 10% of the adult population suffers from hypertension. Renal vascular stenosis is the cause of this hypertension in a subgroup of the patients. This subgroup constitutes 1% of the total hypertensive population. A rise in plasma prorenin often precedes the onset of vascular injury in patients with diabetes mellitus. Plasma prorenin measurements may be useful for predicting which patients will develop vascular injury and for monitoring the progression of the disease.

Human chorionic gonadotropin (hCG) stimulation of the ovaries leads to elevated serum prorenin levels. Prorenin levels, like hCG, are high during the first trimester of pregnancy and decrease in the 2nd and 3rd trimesters. Since hCG levels are increased in Downs syndrome pregnancies relative to normal pregnancies and hCG stimulation leads to increased prorenin levels, this led Applicant to postulate that prorenin (or renin) may also be increased in Downs Syndrome pregnancies.

Accordingly, it would be desirable to provide assay methods and compositions for leptin and/or prorenin and/or renin which would have predictive value with respect to the likelihood that a pregnant woman is carrying a fetus having Downs Syndrome.

SUMMARY OF THE INVENTION

Leptin levels in maternal biological samples are 3-fold higher during pregnancy and correlate positively with human chorionic gonadotropin (hCG) and progesterone levels. hCG levels are increased in Downs syndrome pregnancies relative to normal pregnancies; these facts provided the impetus to the Applicant to determine if the leptin levels correlation with hCG levels may extend to a relative increase in leptin in Downs Syndrome affected pregnancies.

In one aspect, the presently claimed subject matter is directed to a method of determining an increased risk of a woman carrying a Downs Syndrome affected fetus. The method comprising the steps of: quantitatively assaying a sample from a pregnant woman for an amount of leptin in the sample, thereby determining the amount of leptin in the sample; and comparing the amount of leptin in the sample from the pregnant woman with an amount of leptin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of leptin in the sample from the pregnant woman with an amount of leptin found in pregnant women carrying a Downs syndrome affected fetus, thereby determining those at an increased risk of carrying a Downs syndrome affected fetus.

In a further aspect of the presently claimed subject matter, the amount of leptin in the sample as determined in a sample from a pregnant woman is below the median amount of leptin found in a pregnant women carrying a Downs syndrome unaffected fetus

In one embodiment of the present invention, the sample from the pregnant women is selected from the group consisting of serum, plasma or urine.

In another embodiment of the presently claimed subject matter, the sample is taken from a pregnant women in either of the first trimester or the second trimester or the third trimester of pregnancy.

In a particular embodiment of the presently claimed subject matter, the quantitative assay of the sample is performed by immunoassay, more particularly a competitive immunoassay or a direct immunoassay. In a particular embodiment, a radioimmunoassay can be used.

In another embodiment of the presently claimed subject matter, an additional step of analyzing at least one additional analyte selected from the group consisting of hCG, unconjugated estriol, alpha-fetoprotein, inhibin, PAPP-A(Pregnancy Associated Plasma Protein A), progesterone, DHEA-S or Leukocyte acid phosphatase is performed.

In yet another embodiment, an additional step of analyzing at least one additional factor predictive of an increased risk of a fetus being affected by Downs syndrome is performed. In a preferred embodiment, an ultrasound result is the additional predictive factor.

Human chorionic gonadotropin (hCG) stimulation of the ovaries leads to elevated serum prorenin levels. Prorenin levels, like hCG, are high during the first trimester of pregnancy and decrease in the 2nd and 3rd trimesters. Since hCG levels are increased in Downs syndrome pregnancies relative to normal pregnancies and hCG stimulation leads to increased prorenin levels, this led Applicant to postulate that prorenin (or renin) may also be increased in Downs Syndrome pregnancies.

In another aspect, the presently claimed subject matter is directed to a method of determining an increased risk of a woman carrying a Downs Syndrome affected fetus. The method comprising the steps of: quantitatively assaying a sample from a pregnant woman for an amount of prorenin in the sample, thereby determining the amount of prorenin in the sample; and comparing the amount of prorenin in the sample from the pregnant woman with an amount of prorenin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of prorenin in the sample from the pregnant woman with an amount of prorenin found in pregnant women carrying a Downs syndrome affected fetus, thereby determining those at an increased risk of carrying a Downs syndrome affected fetus.

In a further aspect of the presently claimed subject matter, the amount of prorenin in the sample as determined in a sample from a pregnant woman is below the median amount of prorenin found in a pregnant women carrying a Downs syndrome unaffected fetus

In one embodiment of the present invention, the sample from the pregnant women is selected from the group consisting of serum, plasma or urine.

In another embodiment of the presently claimed subject matter, the sample is taken from a pregnant women in either of the first trimester or the second trimester or the third trimester of pregnancy.

In a particular embodiment of the presently claimed subject matter, the quantitative assay of the sample is performed by immunoassay, more particularly a competitive immunoassay or a direct immunoassay. In a particular embodiment, a radioimmunoassay can be used.

In another embodiment of the presently claimed subject matter, an additional step of analyzing at least one additional analyte selected from the group consisting of hCG, unconjugated estriol, alpha-fetoprotein, inhibin, PAPP-A, progesterone, DHEA-S or Leukocyte acid phosphatase is performed.

In yet another embodiment, an additional step of analyzing at least one additional factor predictive of an increased risk of a fetus being affected by Downs syndrome is performed. In a preferred embodiment, an ultrasound result is the additional predictive factor.

In another aspect, the presently claimed subject matter is directed to a method of determining an increased risk of a woman carrying a Downs Syndrome affected fetus. The method comprising the steps of: quantitatively assaying a sample from a pregnant woman for an amount of renin in the sample, thereby determining the amount of renin in the sample; and comparing the amount of renin in the sample from the pregnant woman with an amount of renin found in pregnant women carrying a Downs syndrome unaffected fetus and comparing the amount of renin in the sample from the pregnant woman with an amount of renin found in pregnant women carrying a Downs syndrome affected fetus, thereby determining those at an increased risk of carrying a Downs syndrome affected fetus.

In still further aspects combinations of leptin and/or prorenin and/or renin are assayed and statistical methods of analyzing the contribution of more than two factors to the likelihood of an outcome, as are known in the art, are used to predict those at an increased risk of carrying a Downs syndrome affected fetus.

Other features and advantages of the invention will become apparent from the following detailed description.