Inventors: Macri; James N. (170 Sidney St., Oyster Bay, NY 11771) Appl. No.: 925844 Filed: August 7, 1992

Primary Examiner: Rosen; Sam Attorney, Agent or Firm: Kenyon & Kenyon United States Patent 5,324,667 June 28, 1994

Parent Case Text
This application is a continuation-in-part of application Ser. No. 07/868,160, filed Apr. 14, 1992, which is a continuation-in-part of application Ser. No. 07/420,775, filed Oct. 12, 1989, which is a continuation-in-part of application 07/360,603, filed Jun. 1, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/349,373, filed May 8, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/311,808 filed Feb. 17, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/297,481, filed Jan. 17, 1989, now abandoned.

Claims
I claim:

1. A screening method for determining a pregnant woman's risk of carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood for free beta (human chorionic gonadotropin (HCG)) level during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; and comparing said level of free beta (HCG) to reference values of the level for free beta (HCG) during the time period in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with Down syndrome, wherein a higher level of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

2. The method of claim 1 further comprising measuring the pregnant woman's maternal blood for alpha-fetoprotein (AFP) level during the time period and incorporating reference values of the levels AFP during the time period of pregnancy in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, into said comparison wherein a lower level of AFP is indicative of a higher probability of risk. is carrying a fetus with Down syndrome comprising: measuring

3. The method of claim 1 wherein the measurement is performed utilizing a biosensor.

4. A method for determining the risk that a pregnant woman is carrying a fetus with Down syndrome comprising: measuring said pregnant woman's maternal blood level of a fragment of free beta (human chorionic gonadotropin (HCG)) during a time period selected from the group consisting of the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy and comparing the measurement of said level of the fragment of free beta (HCG) to reference data containing reference values of the level of the fragment of free beta (HCG) during the time period in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of the pregnant woman's risk wherein a higher level of the fragment of free beta (HCG) is indicative of a higher probability of carrying a fetus with Down syndrome.

5. The method of claim 4 wherein the fragment of free beta (HCG) is selected from the group consisting of: the protein portion of free beta (HCG; the carbohydrate portion of free beta (HCG); and the portion of free beta (HCG) located at about the junction of the carbohydrate and the protein portions of free beta (HCG).

6. The method of claim 5 further comprising measuring the pregnant woman's maternal blood for alpha-fetoprotein (AFP) level during the time period and incorporating reference values of the levels AFP during the time period in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, into said comparison wherein a lower level of AFP is indicative of a higher probability of risk.

7. A method for determining whether a pregnant woman's risk of carrying a fetus with Down syndrome warrants further testing comprising: measuring said pregnant woman's maternal blood level of an analyte during a time period selected from the group consisting of the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; using an assay for free beta (HCG) and comparing said level of said analyte to a set of reference data containing reference values at various gestational ages of the level of the analyte during the time period in pregnant women carrying Down syndrome fetuses and pregnant women carrying normal fetuses, said comparison being indicative of pregnant woman's risk of carrying a fetus with Down syndrome wherein a higher level of the analyte is indicative of a higher probability of carrying a fetus with Down syndrome.

8. The method of claim 1, 2, or 7 wherein the free beta (HCG) is an aberrant form of free beta (HCG).

9. The method of claim 8 wherein the free beta (HCG) is nicked free beta (HCG).

10. A screening method for determining a pregnant woman's risk of carrying a fetus with Down syndrome comprising: measuring said pregnant woman's urine level of free beta (human chorionic gonadotropin (HCG)) level during a time period selected from the group consisting of: the first trimester of pregnancy; the second trimester of pregnancy and the third trimester of pregnancy; and comparing said level of free beta (HCG) to reference value of the level for free beta (HCG) in: (1) pregnant women carrying Down syndrome fetuses and (2) pregnant women carrying normal fetuses, said comparison being indicative of said pregnant woman's risk of carrying a fetus with Down syndrome.

Description
FIELD OF THE INVENTION

The present invention relates to a method for detecting fetal Down syndrome (Trisomy 21), trisomy 13, trisomy 18, Turners syndrome and other chromosomal anomalies during prenatal screening. More particularly the present invention relates to a method for improving detection efficiency in prenatal screening for Down syndrome by measuring the amount of free beta (human chorionic gonadotropin "HCG") and nicked or fragmented or aberrant forms of free beta (HCG) all of which are referenced throughout this application as free beta (HCG).

BACKGROUND OF THE INVENTION

Down syndrome, also referred to as Trisomy 21, is the most common congenital cause of severe mental retardation. Generally, fetal Down syndrome can be determined by diagnostic procedures including amniocentesis or chorionic villus sampling and karyotyping. However, these diagnostic procedures are invasive and involve risk to the woman and the fetus. For this and other reasons, amniocentesis or chorionic villus sampling and karyotyping are not routinely performed during all pregnancies. Instead, one or more screening methods may be utilized to determine when the risk to the pregnancy warrants the risk of undergoing an invasive diagnostic procedure.

The incidence of Down syndrome increases significantly with increasing maternal age. Historically, the prenatal detection of Down syndrome has focused on pregnant women at and over the age of 35, at which ages the risks of Down syndrome approach or exceed the risks of diagnostic procedures utilized to detect fetal Down syndrome. Therefore the standard method of prenatal screening has involved selecting women for diagnostic amniocentesis on the basis of maternal age. Age, however, is an inadequate screening criterion in that only about 20% of all Down syndrome pregnancies can be detected by carrying out amniocentesis and karyotyping on the 5% of pregnant women most at risk, that is, those aged 35 years or greater. And, because in actual clinical practice only about half of the women aged 35 years or greater undergo amniocentesis and karyotyping, fewer than 10% of Down syndrome pregnancies are prenatally detected.

In 1984 an association between lowered maternal blood alphafetoprotein (AFP) levels and fetal Down syndrome was discovered. For example, see "An association between low maternal serum alpha-fetoprotein and fetal chromosomal abnormalities"; Merkatz, Macri, et al.; Am. J. Obstet. Gynecol. 148:996, 1984; the disclosure of which is hereby incorporated by reference. In this publication it was noted that other chromosomal trisomies, in particular Trisomy 13 and Trisomy 18, were also associated with lowered maternal blood AFP levels. The incidence of these additional chromosomal trisomies (1 in 5000 pregnancies and 1 in 6600 pregnancies, respectively) is significantly lower than the general a priori risk associated with Trisomy 21 (Down syndrome, 1 in 800 pregnancies). However, because of the association of these other chromosomal trisomies with lowered MSAFP levels, and elevated or depressed free beta (HCG) levels, such abnormalities will also be detected within a screening protocol utilizing maternal blood AFP and free beta (HCG) and possibly additional markers described herein. It is obvious to those skilled in the art that in using the protocol described herein for Trisomy 21, the detection of Trisomy 13, Trisomy 18, Turners syndrome and other chromosomal anomalies may also be accomplished.

The association between lowered maternal blood AFP levels and fetal Down syndrome presented the opportunity to use a non-invasive blood screening test in the detection of Down syndrome cases in young, apparently unaffected families where approximately 80% of Down syndrome cases occur. It is estimated that the use of a screening test based on low maternal blood AFP (as a screening marker) would lead to the prenatal detection of approximately 20% of all cases of fetal Down syndrome.

Another method for screening-involves measuring the level of unconjugated estriol (UE) in maternal blood. For example, see "Maternal blood screening for Dwon syndrome in early pregnancy"; Wald, et al. British Journal of Obstetrics and Gynocology (BMJ) Volume 95, April 1988, the disclosure of which is hereby incorporated by reference.

More recently an association between elevated maternal blood levels of the Intact HCG molecule and the alpha subunit of HCG (HCG is composed of two subunits) and fetal Down syndrome was discovered. For example, see "Abnormal Maternal Serum Chorionic Gonadotropin Levels in Pregnancies with Fetal Chromosome Abnormalities"; Bogart, Pandian and Jones; Prenatal Diagnosis, Vol. 7, 623-630 (1987) the disclosure of which is hereby incorporated by reference. In the Bogart article it is estimated that the use of elevated maternal blood Intact HCG levels and elevated maternal blood levels of the alpha subunit of HCG, would detect approximately 68% of the chromosomally abnormal fetuses. However, these results were obtained from a study on pregnancies at 18-25 weeks of gestation and the affected cases appear to be of women previously identified as being at risk for Down syndrome.

U.S. Pat. No. 4,874,693 to Bogart discloses an association between elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG, in weeks 18-25 of pregnancy and fetal Down syndrome. In the Bogart patent it is estimated that the use of elevated maternal blood HCG levels and elevated maternal blood levels of the alpha subunit of HCG in a screening protocol, would detect a greater percentage of chromosomally abnormal fetuses than the use of AFP or UE alone. In a paper entitled "Human Chorionic Gonadotropin Levels in Pregnancies with Aneuploid Fetuses" (Bogart et al., Prenatal Diagnosis, Vol. 9, 379-384 (1989)), Bogart discloses that a screening method utilizing HCG and the alpha subunit of HCG is not useful at 9-11 weeks gestation (the first trimester of pregnancy) for selecting pregnancies at risk for fetal aneuploidy (including Down syndrome).

Generally, as suggested above, screening by evaluation of maternal blood HCG has involved only the measurement of the Intact HCG molecule and additionally the measurement of the alpha subunit of HCG. Although these screening methods do detect fetal Down syndrome, there is a need and a desire for a method which detects a greater percentage of fetal Down syndrome cases.

I have discovered a previously unknown association between elevated levels of maternal blood free beta (HCG) and fetal Down syndrome. I have also discovered a previously unknown association between the maternal blood level of free beta (HCG) and the maternal blood level of AFP and fetal Down syndrome. I have further discovered a previously unknown association between the ratio of the maternal blood level of free beta (HCG) to the maternal blood level of the intact HCG molecule and fetal Down syndrome. I have still further discovered that using a multivariate discriminant analysis technique improves the detection efficiency of a screening method using the maternal blood level of free beta (HCG), or the maternal blood level of free beta (HCG) and the maternal blood level of AFP, or the log of either, or the log of both, especially when gestational age is also incorporated as a variable in the discriminant analysis technique, for a chosen risk cut-off level. Gestational age refers to the age of the pregnant woman's fetus. Detection efficiency refers to the percentage of cases of fetal Down syndrome which are correctly detected for a chosen risk cut off level. The risk cut off level will be more fully explained in a following section. Discriminant analysis is a generally known approach to multivariate analysis involving the separation of a population into two or more groups on the basis of probability. Discriminant analysis is also sometimes described as a way of constructing a linear combination of independent variables, thus reducing the problem of measuring group differences to a univariate problem. A general discussion of discriminant analysis can be found in Marketing Research; Churchill, G.A.; Dryden, 1976; Chapter 15, pages 530-543, the disclosure of which is hereby incorporated by reference. I have discovered that subjecting the maternal blood levels of free beta (HCG), the maternal blood levels of intact HCG, the ratio of the maternal blood level of free beta (HCG) to the maternal blood level of the intact HCG molecule, the maternal blood level of AFP, the maternal blood level of UE, and gestational age to multi-variate discriminant analysis detects a greater percentage, with a lower false positive rate, of fetal Down syndrome cases than any other known screening method for the prenatal detection of Down syndrome. I have further discovered that a still greater number the cases of fetal Down syndrome may be detected by using only the measurements of the maternal blood levels of free beta (HCG) and the maternal blood levels of AFP and subjecting the log of each measurement and gestational age to a multivariate discriminant analysis. These and other discoveries will be more fully explained in the Summary of the Invention section and the Detailed Description of the Invention section.

One object of the present invention is to provide a method and process for screening for fetal Down syndrome which detects a greater percentage of fetal Down syndrome cases with a given false positive rate than other known prenatal screening methods.

Another object of the present invention is to provide a method and process for screening for fetal Down syndrome which has a lesser false positive rate for a given detection percentage than other known methods.

A still further object of the present invention is to apply multi-variate discriminant analysis to methods for screening for Down syndrome to detect a greater percentage of fetal Down syndrome cases with a lesser false positive rate.

A further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the level of maternal blood free beta (HCG).

A still further object of the present invention is to provide a method and process for screening for fetal Down syndrome by measuring the maternal blood level of AFP and the maternal blood level of free beta (HCG).

Other objects and advantages of the present invention will become apparent in the following description of the invention.

SUMMARY OF THE INVENTION

To achieve these and other objects, according to the present invention a pregnant woman's (hereinafter sometimes referred to as the patient) maternal blood levels of free beta (HCG) are measured by conventional immunological techniques which can include immunoassay techniques such as those referred to in the papers above, and other techniques known in the art. The level of free beta (HCG) is then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. To improve detection efficiency, the level of free beta (HCG) and the gestational age can be compared to a set of reference data. To further improve detection efficiency, a patient's maternal blood levels of free beta (HCG) and AFP (referred to as "markers") are measured by conventional immunological methods, including assay techniques known to the art such as those referred to in the papers above. The levels of each marker are then compared to a set of reference data to determine the patient's risk of carrying a fetus with Down syndrome. A multivariate discriminant analysis technique is advantageously used to compare the levels of the markers to a set of reference data. More particularly, a patient specific risk is then calculated using Bayes rule, the patient's a priori risk, and the relative frequencies for unaffected and affected pregnancies which are determined by incorporating the log of the patient's quantitative levels of each marker into the probability density functions for the reference data developed using multivariate discriminant analysis. If the patient's risk of carrying a fetus with Down syndrome is greater than a given risk cut-off level, the patient should be counseled about further diagnostic tests to confirm the presence of Down syndrome.

Similarly, if the method of the present invention is utilized in a screening protocol for trisomy 13, trisomy 18, Turner's syndrome or other chromosomal anomalies, the patient's risk of carrying a fetus with the anomaly may be determined using a multivariate discriminant analysis technique whereby the level of free beta (HCG) and AFP are compared to a set of reference data.

Incorporating gestational age as a marker along with the level of free beta (HCG) and the level of AFP will further improve detection efficiency. Since the maternal blood level of free beta (HCG) and the maternal blood level of AFP for a number of samples tend to be distributed according to a log-gaussian distribution curve, the greatest detection efficiency can be achieved by incorporating the log of the patient's quantitative levels of each marker and gestational age into the probability density functions for the reference data developed using multivariate discriminant analysis.

As described herein, and in my prior applications, free beta (HCG) can exist in many forms including intact free beta (HCG) and so called aberrant forms of free beta (HCG). These aberrant forms of free beta (HCG) may include fragments of the complete free beta (HCG) molecule and "nicked" free beta (HCG). Additional details concerning these forms, and others, of free beta (HCG) is contained in the following sections. Immunological methods that measure any or all of the forms of free beta (HCG) fall within the scope of the present invention.

The conventional immunological techniques suitable for use in the present invention also include the use of biosensors. A biosensor generally comprises a biologically-derived sensing element, such as an antibody, linked to a transducer that can produce an electrical signal or other observable readout. When a certain amount of the substance to be measured, for example free beta (HCG) or a form thereof, contacts the sensing element a reaction takes place and the transducer translates the reaction into a change of color, fluorecence, temperature, electrical current or other electro-chemical signal. A biosensor that measures the level of free beta (HCG) and or a form of free beta (HCG) is within the scope of the method of the present invention.

An advantage of the method and process of the present invention is that it correctly predicts a higher percentage of fetal Down syndrome cases, with a lesser false positive rate than other known methods and processes.