Molecular pathology selected abstracts

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Next-generation sequencing of circulating cell-free DNA during pregnancy

Non-invasive, next-generation sequencing-based testing of cell-free DNA derived from maternal plasma for fetal chromosomal anomalies is increasingly being adopted in medical practice. In most cases, plasma cell-free DNA (cfDNA) obtained from pregnant women originates in the placenta or is derived from maternal cells. The fetal fraction (placental origin cfDNA:total cfDNA) reaches a threshold for effective analysis at approximately 10 weeks of gestation. Sequencing information, most commonly pertaining to chromosomal copy number changes, is compared to reference datasets derived from profiling pregnant women carrying euploid fetuses to screen for chromosomal aneuploidy. Prenatal cfDNA testing has gone from a research setting to transforming prenatal care worldwide in less than a decade. As of late 2017, between 4 and 6 million pregnant women had undergone analysis of DNA from their plasma to screen for fetal aneuploidy. CfDNA testing is primarily used to detect trisomy 13, 18, and 21. In some studies, estimates of analytical sensitivity and specificity in high-risk women range from 93 to 99.9 percent, which is superior to traditional testing involving a combination of serum biochemical assays and sonographic metrics. Importantly, low false-positive rates have contributed to reducing the number of invasive confirmatory procedures, such as amniocentesis and chorionic villus sampling, by as much as 40 to 76 percent, according to some studies. Increased testing volume has expanded the medical community’s understanding of the limitations of such testing. For instance, documented causes of false-positive results in cfDNA aneuploidy testing have been found in maternally derived cfDNA (chromosomal abnormality, cancer), placentally derived cfDNA (confined placental mosaicism, death of a twin in utero), and allograft derived cfDNA (bone marrow transplant). Furthermore, medical conditions such as obesity and thromboembolic disorders, which are associated with a low fetal fraction, can lead to false-negative test results. While cfDNA aneuploidy screening has been adopted as a first-tier test for all pregnant women in some European countries, numerous barriers have prevented more widespread adoption in the United States. These barriers include variable reimbursement by insurers and public payers, an education gap among health care providers, and a lack of resources for required pretest counseling. The screening is monitored by the Clinical Laboratory Improvement Amendments (CLIA) program as a laboratory-developed test validated by independent laboratories. No FDA-approved cfDNA test is available. In addition to aneuploidy screening, cfDNA testing is clinically available for evaluating microdeletion and microduplication syndromes, such as terminal 5p deletion in Cri du chat syndrome. Avenues for expanding such testing include single-gene assessments, such as for rhesus D genotyping in rhesus D-negative women, skeletal dysplasias, beta-thalassemia, sickle cell anemia, and hemophilia.

Bianchi DW, Chiu RWK. Sequencing of circulating cell-free DNA during pregnancy. N Engl J Med. 2018;379(5):464–473.

Correspondence: Dr. Diana Bianchi at diana.bianchi@nih.gov