Circulating nucleic acids for prenatal diagnostics

Background

Prenatal diagnosis is becoming an essential part of prenatal care for a growing number of patients. To make a definitive diagnosis, traditional methods of sampling fetal genetic material, such as chorionic villus sampling and amniocentesis, are invasive and introduce a risk of spontaneous miscarriage. The discovery of cell-free fetal DNA (cffDNA) in the circulation of pregnant women led to the possibility of noninvasive genetic and chromosomal assessment of the fetus through the sampling of maternal peripheral blood. The clinical introduction of cffDNA tests has led to a substantial reduction in the number of invasive procedures performed worldwide.

Content

This chapter describes the biological properties of circulating cffDNA, the applications that have been developed, and their clinical uses. Additionally, analytical characteristics of cffDNA testing are highlighted. Circulating cffDNA is derived from placental cell turnover. This DNA from apoptotic placenta cells is highly fragmented, with the majority of fragments less than 200 bp in length. Circulating cffDNA exists with a substantial background of maternal DNA. Circulating cffDNA can be detected from early pregnancy onward, and it rapidly (about 1 hour half-life) disappears from maternal circulation following delivery of the newborn. The analysis of cffDNA is now clinically used for the assessment of sex-linked diseases, fetal blood group incompatibility, fetal chromosomal aneuploidies, and some single-gene diseases. Analytical protocols are designed to maximize the yield of fetal DNA by minimizing maternal DNA contamination and by preserving the abundance of short DNA molecules. The inclusion of internal positive controls for the presence of fetal DNA or the measurement of fetal DNA fraction is an important quality control parameter.

Maternal age

The probability of giving birth to an infant with Down syndrome increases with advancing maternal age. The risk of giving birth to an affected infant at term is estimated to be less than 1 in 1000 at a maternal age of 29 years and younger, but it increases to 1 in 385 at 35 years of age. Hence, prior to the development of more elaborate prenatal screening strategies, it was customary to offer prenatal diagnosis to women aged 35 years or older. However, because a significant proportion of women become pregnant before 35 years of age, maternal age alone would only identify 51% of Down syndrome–affected pregnancies at a 14% false-positive rate.

Serum biochemistry screening

The combination of maternal age assessment with maternal serum screening of various biomarkers between 15 and 22 weeks of gestation was later developed as a second trimester screening protocol to identify high-risk pregnancies. This screening strategy is referred to as the “triple test,” and the serum biomarkers include alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol. When the analytical cutoff values are set to give a 5% false-positive rate, the detection sensitivity for Down syndrome is 70%. Testing maternal serum inhibin A and the triple test markers during the second trimester, termed the “quadruple test,” provided a detection sensitivity of 75% at a false-positive rate of 5%.

First trimester screening

While the triple and quadruple tests are used during the second trimester, alternative Down syndrome screening strategies have been developed for testing during the first trimester. One of these strategies for first trimester Down syndrome screening measures free β-human chorionic gonadotropin and pregnancy-associated plasma protein A. ^, Down syndrome is associated with an increase in fetal nuchal translucency measured by first trimester ultrasound. Subsequently, the combination of first trimester biochemical markers, fetal nuchal translucency, and maternal age assessments came to be known as the “first trimester combined test.” With a false-positive rate of 5%, the test could detect 95% of Down syndrome fetuses.

The approaches described above have been incorporated into many prenatal screening programs. However, the main disadvantage of these tests lies in their high false-positive rates. Most of the test cutoff values used to identify those deemed to be at high risk had false-positive rates of 5%. This meant that 1 in every 20 women would be labeled as high risk and would need to face the decision of whether or not to undergo an invasive diagnostic procedure. Because the average Down syndrome risk is 1 in 800, this meant that a substantial number of women undergoing an invasive diagnostic procedure did not carry an affected fetus. Therefore there was a need to identify or develop more robust screening methods that had lower false-positive rates and improved detection rates.