Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing

Judith Walsh, MD, MPH



In This Article


Chromosomal Aneuploidy

Prenatal testing is widely recommended and widely used to screen for chromosomal abnormalities. Down syndrome (trisomy 21), Edwards syndrome (Trisomy 18) and Patau syndrome (Trisomy 13) are three aneuploid disorders with significant consequences which can be detected by prenatal evaluation.

Down syndrome (Trisomy 21) is the most common chromosome abnormality in live births. Down syndrome accounts for the commonest form of intellectual disability caused by a known chromosome problem. Individuals with Down syndrome have a learning disability that is moderate to severe (average IQ of 50), characteristic facial features, short stature, cardiac and intestinal defects, problems with vision hearing and increased infection risk. There is significant morbidity and mortality in affected individuals as well as adding a psychosocial and financial burden to the family.

The prevalence of Down syndrome is overall about 1 in 629 births.[1] However the prevalence increases with increasing maternal age, so that the risk is about one in 1,340 at age 25; one in 353 at age 35 and in 35 at age 45.[2,3]

Trisomy 18 (Edwards syndrome) is the second most common autosomal trisomy. The incidence is about 1 in 5,500 live births, but the risk increases with increasing maternal age. About half of babies born with Trisomy 18 die within the first week of life and only five to ten percent survive to one year. Those who do survive have intellectual disability.[4]

Trisomy 13 or Patau syndrome is another chromosomal trisomy where 80% of infants die within the first month. Those who survive have intellectual disability, seizures and failure to thrive.[4]

Prenatal Diagnostic Testing

The American College of Obstetricians and Gynecologists (ACOG) recommends that pregnant women be offered screening for chromosomal abnormalities, regardless of maternal age.[5] There are multiple options for noninvasive prenatal screening for fetal chromosomal abnormalities. The noninvasive tests typically include measurement of maternal serum markers which are interpreted in the context of maternal age and many also include ultrasound findings (nuchal translucency-ultrasound measurement of the back of the neck).

The first trimester combined test includes nuchal translucency (NT), crown-rump length as well as pregnancy associated plasma protein-A (PAPP-A) and free or total beta human chorionic gonadotropin (beta-HCG). This screening test can be performed in the first trimester (before 13 weeks).

A second category of noninvasive tests are the "integrated tests" which require maternal plasma analytes during both the first and the second trimesters. These include additional markers such as alpha fetoprotein (AFP), unconjugated estriol (uE3) and inhibin A. The full integrated test has the highest detection rate for Down syndrome and the lowest screen positive rate, but the disadvantage is that the testing cannot be completed until the second trimester.

The results of the noninvasive tests can either be "screen negative" or "screen positive." A screen negative result means that the patient's risk of having a baby with Down syndrome is less than some predetermined level. The patient is provided with the actual risk (eg risk for Down Syndrome is 1/500). A screen positive result means that the risk for Down syndrome is at or above the cut off point. A patient who has a "screen positive" result is offered a diagnostic test, either chorionic villus sampling (CVS) or amniocentesis. ACOG recommends that all patients should be provided with an individualized risk acknowledging that different patients have different concepts of what would be considered a personal positive result.

In summary, for the currently available noninvasive tests, despite the various proposed combinations of noninvasive tests, existing screening methods have detection rates of 90–95% and false positive rates of 3–5%, but all offer a combination of noninvasive testing followed by an offer of diagnostic testing if the screening test is positive.[6–9]

Diagnostic Tests

The currently available diagnostic tests are amniocentesis and CVS. Both have the ability to definitely show fetal karyotyping. CVS has the advantage of being able to be performed earlier in pregnancy (during the first trimester), but is associated with a small risk of pregnancy loss. Amniocentesis cannot be performed until later in the pregnancy, (often after a woman is already obviously pregnant) and is also associated with a small risk of miscarriage. Both of these tests provide actual chromosomal analyses and can either confirm or disconfirm an abnormal screening test.

Cell Free Fetal DNA (cfDNA)

Prenatal diagnosis can use cell-free fetal nucleic acids in maternal blood to detect fetal chromosomal abnormalities. Intact fetal cells can be found in the maternal circulation, leading to the possibility that maternal blood could be used to diagnose fetal disease. Because the absolute amount of fetal cells relative to maternal blood is very small, they can be difficult to detect. In addition, cells from prior pregnancies can persist in the maternal circulation, which further limits the use of actual fetal cells.

However, cell free nucleic acids (DNA and RNA) are more plentiful in the maternal circulation. In addition, fetal DNA has a very short half life; thus any fetal DNA that is found is due to the current pregnancy. Fetal DNA can often be detected by the fifth postmenstrual week and can almost always be detected by the 9th postmenstrual week.[10] Fetal DNA is approximately three to six percent of total DNA in the maternal circulation. The percentage of fetal DNA increases with increasing gestational age.[11]

One of the main challenges of the use of cfDNAfor diagnosis is differentiating the fetal DNA from the maternal DNA. One of the best methods is to identify DNA sequences only found on the Y chromosome, since normal women do not have Y chromosomes. Another way is to identify DNA sequences which are associated with paternally inherited fetal conditions, since normal women would not be expected to have them. Finding maternally linked alleles is potentially more difficult, since it would be harder to differentiate maternal and fetal DNA. Strategies that have been used include finding fetal DNA that is methylated differently than maternal DNA or looking at differences in DNA length.[12,13] Fetal DNA fragments are shorter than maternal DNA fragments; however this difference has not proven useful in differentiating the two types of DNA.[14]

There are at least two techniques for isolating fetal DNA from maternal DNA. Massively parallel signature sequencing (MPSS) is a random analysis of millions of cfDNA fragments. This technique sequences short segments of cfDNA from the mother and the fetus and assigns them to specific chromosomes. The number of chromosome counts are then compared to a control value of other chromosomes and if there is an excess of a particular chromosome (e.g. 21), trisomy is suspected. This technique requires analysis of a very large number of DNA fragments per sample – estimates are about 25 million, which could potentially limit its clinical utility.

The other technique is directed DNA analysis. The goal of directed DNA analysis is to selectively sequence relevant chromosomes. Digital analysis of selected regions (DANSR) is a recently developed process of analyzing counts form assays targeted against selected genomic regions on particular chromosomes.[15] This technique is touted as being potentially more efficient than MPSS because it uses fewer genetic fragments. Both of these techniques are currently being evaluated for use in noninvasive prenatal testing.

The potential role of cfDNA in prenatal diagnosis would be either as a primary screening test (replacing the currently available screening tests) or as a "secondary screening test." Since it is not a diagnostic test, confirmation of positive results by a diagnostic test would still be required. However, the potential advantage of using cfDNA as a secondary screening test would be to reduce the number of invasive procedures and the resulting loss of normal fetuses. Thus CTAF is evaluating the role of cfDNA as a primary or secondary screening test for fetal aneuploidy.