Non-invasive prenatal diagnosis

Details Non-invasive prenatal diagnosis Non-invasive prenatal diagnosis

1999-11-29

2001-07-10

Arthur, Lisa B. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091500, C435S440000

Reexamination Certificate

active

06258540

ABSTRACT:

This invention relates to prenatal detection methods using non-invasive techniques. In particular, it relates to prenatal diagnosis by detecting foetal nucleic acids in serum or plasma from a maternal blood sample.
BACKGROUND OF THE INVENTION
Conventional prenatal screening methods for detecting foetal abnormalities and for sex determination traditionally use foetal samples derived by invasive techniques such as amniocentesis and chorionic villus sampling. These techniques require careful handling and present a degree of risk to the mother and to the pregnancy.
More recently, techniques have been devised for predicting abnormalities in the foetus and possible complications in pregnancy, which use maternal blood or serum samples. Three markers commonly used include alpha-foetoprotein (AFP—of foetal origin), human chorionic gonadotrophin (hCG) and estriol, for screening for Down's Syndrome and neural tube defects. Maternal serum is also currently used for biochemical screening for chromosomal aneuploidies and neural tube defects. The passage of nucleated cells between the mother and foetus is now a well-recognised phenomenon (Lo et al 1989; Lo et al 1996). The use of foetal cells in maternal blood for non-invasive prenatal diagnosis (Simpson and Elias 1993) avoids the risks associated with conventional invasive techniques. WO 91/08304 describes prenatal genetic determination using foetal DNA obtained from foetal cells in the maternal blood. Considerable advances have been made in the enrichment and isolation of foetal cells for analysis (Simpson and Elias 1993; Cheung et al 1996). However, these techniques are time-consuming or require expensive equipment.
Recently, there has been interest in the use of plasma or serum-derived DNA for molecular diagnosis (Mulcahy et al 1996). In particular, it has been demonstrated that tumour DNA can be detected by the polymerase chain reaction (PCR) in the plasma or serum of some patients (Chen et al 1996; Nawroz et al 1996).
GB 2 299 166 describes non-invasive cancer diagnosis by detection of K-ras and N-ras gene mutations using PCR-based techniques.
SUMMARY AND OBJECTS OF THE INVENTION
It has now been discovered that foetal DNA is detectable in maternal serum or plasma samples. This is a surprising and unexpected finding; maternal plasma is the very material that is routinely discarded by investigators studying non-invasive prenatal diagnosis using foetal cells in maternal blood. The detection rate is much higher using serum or plasma than using nucleated blood cell DNA extracted from a comparable volume of whole blood, suggesting that there is enrichment of foetal DNA in maternal plasma and serum. In fact, the concentration of foetal DNA in maternal plasma expressed as a % of total DNA has been measured as from 0.39% (the lowest concentration measured in early pregnancy), to as high as 11.4% (in late pregnancy), compared to ratios of generally around 0.001% and up to only 0.025% for cellular fractions (Hamada et al 1993). It is important that foetal DNA is found in maternal plasma as well as serum because this indicates that the DNA is not an artefact of the clotting process.
This invention provides a detection method performed on a maternal serum or plasma sample from a pregnant female, which method comprises detecting the presence of a nucleic acid of foetal origin in the sample. The invention thus provides a method for prenatal diagnosis.
The term “prenatal diagnosis” as used herein covers determination of any maternal or foetal condition or characteristic which is related to either the foetal DNA itself or to the quantity or quality of the foetal DNA in the maternal serum or plasma. Included are sex determination, and detection of foetal abnormalities which may be for example chromosomal aneuploidies or simple mutations. Also included is detection and monitoring of pregnancy-associated conditions such as pre-eclampsia which result in higher or lower than normal amounts of foetal DNA being present in the maternal serum or plasma. The nucleic acid detected in the method according to the invention may be of a type other than DNA e.g. mRNA.
The maternal serum or plasma sample is derived from the maternal blood. As little as 10 &mgr;l of serum or plasma can be used. However it may be preferable to employ larger samples in order to increase accuracy. The volume of the sample required may be dependent upon the condition or characteristic being detected. In any case, the volume of maternal blood which needs to be taken is small.
The preparation of serum or plasma from the maternal blood sample is carried out by standard techniques. The serum or plasma is normally then subjected to a nucleic acid extraction process. Suitable methods include the methods described herein in the examples, and variations of those methods. Possible alternatives include the controlled heating method described by Frickhofen and Young (1991). Another suitable serum and plasma extraction method is proteinase K treatment followed by phenol/chloroform extraction. Serum and plasma nucleic acid extraction methods allowing the purification of DNA or RNA from larger volumes of maternal sample increase the amount of foetal nucleic acid material for analysis and thus improve the accuracy. A sequence-based enrichment method could also be used on the maternal serum or plasma to specifically enrich for foetal nucleic acid sequences.
An amplification of foetal DNA sequences in the sample is normally carried out. Standard nucleic acid amplification systems can be used, including PCR, the ligase chain reaction, nucleic acid sequence based amplification (NASBA), branched DNA methods, and so on. Preferred amplification methods involve PCR.
The method according to the invention may be particularly useful for sex determination which may be carried out by detecting the presence of a Y chromosome. It is demonstrated herein that using only 10 &mgr;l of plasma or serum a detection rate of 80% for plasma and 70% for serum can be achieved. The use of less than 1 ml of maternal plasma or serum has been shown to give a 100% accurate detection rate.
The method according to the invention can be applied to the detection of any paternally-inherited sequences which are not possessed by the mother and which may be for example genes which confer a disease phenotype in the foetus. Examples include:
a) Foetal rhesus D status determination in rhesus negative mothers (Lo et al 1993). This is possible because rhesus D positive individuals possess the rhesus D gene which is absent in rhesus D negative individuals. Therefore, the detection of rhesus D gene sequences in the plasma and serum of a rhesus D negative mother is indicative of the presence of a rhesus D positive foetus. This approach may also be applied to the detection of foetal rhesus D mRNA in maternal plasma and serum.
b) Haemoglobinopathies (Camaschella et al 1990). Over 450 different mutations in the beta-globin gene have been known to cause beta-thalassaemia. Provided that the father and mother carry different mutations, the paternal mutation can be used as an amplification target on maternal plasma and serum, so as to assess the risk that the foetus may be affected.
c) Paternally-inherited DNA polymorphisms or mutations. Paternally-inherited DNA polymorphisms or mutations present on either a Y or a non-Y chromosome, can be detected in maternal plasma and serum to assess the risk of the foetus being affected by a particular disease by linkage analysis. Furthermore, this type of analysis can also be used to ascertain the presence of foetal nucleic acid in a particular maternal plasma or serum sample, prior to diagnostic analysis such as sex determination. This application will require the prior genotyping of the father and mother using a panel of polymorphic markers and then an allele for detection will be chosen which is present in the father, but is absent in the mother.
The plasma or serum-based non-invasive prenatal diagnosis method according to the invention can be applied to screening for Down's Syndrome and other chromosomal an

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