Most fetuses with structural abnormalities have a normal karyotype and a normal CMA result. Whole genome sequencing (WGS) is a technique for analyzing the entire genome. Whole exome sequencing (WES) analyzes just the DNA coding regions, which account for approximately 1 percent of the genome. These next-generation sequencing tools are increasingly used in the postnatal setting to evaluate suspected genetic syndromes and intellectual disability. The American College of Medical Genetics Board of Directors (2012) states that WGS and WES may be considered for evaluation of the fetus with a likely genetic disorder in which CMA has failed to arrive at a diagnosis. The American College of 0bstetricians and Gynecologists (201i6a) suggests that this be in only selected circumstances, for example, with recurrent or lethal anomalies in which other approaches have been noninformative. Importantly, WGS and WES have signiicant limitations in their current form, including turnaround times that may be prohibitively long and a high rate of variants of uncertain signiicance (American College of �1edical Genetics, 2012; Atwal, 2014). As a result, the clinical utility of this promising technology for prenatal cases is currently limited. • Fetal DNA in the Maternal Circulation Fetal cells are present in maternal blood at a very low concentration, only 2 to 6 cells per milliliter (Bianchi, 2006). Sometimes, intact fetal cells may persist in the maternal circulation for decades following delivery. Persistent fetal cells may engraft in the mother and result in microchimerism, which has been implicated in maternal autoimmune diseases such as scleroderma, systemic lupus erythematosus, and Hashimoto thyroiditis. For prenatal diagnosis, the use of intact fetal cells from maternal blood is limited by low cell concentration, cell persistence into successive pregnancies, and diiculties in distinguishing fetal from maternal cells. In these cases, however, cell-free DNA overcomes these limitations. Cell-Free DNA hese DNA fragments are derived from maternal cells and from apoptotic placental trophoblast cells-although DNA from the latter is often termed "fetal." Cell-free DNA can be reliably genotyping Maternal Real-time quantitative PCR Detection of single-gene disorders Genetics 273 detected in maternal blood after 9 to 10 weeks' gestation (American College of 0bstetricians and Gynecologists, 20 17b). he proportion of cell-free DNA that is placental is called the fetal fraction, and it composes approximately 10 percent of the total circulating cell-free DNA in maternal plasma. Unlike intact fetal cells, cell-free DNA is cleared within minutes from maternal blood. In research settings, cell-free DNA has been used to detect numerous single-gene disorders transmitted through paternally inherited alleles. These include myotonic dystrophy, achondroplasia, Huntington disease, congenital adrenal hyperplasia, cystic fibrosis, and a-thalassemia (Wright, 2009). Clinical applications of cell-free DNA are aneuploidy screening, fetal sex determination, and h D genotyping (Fig. 13-14). Aneuploidy Screening. Several diferent types of assays are used to screen for fetal autosomal trisomies and sex chromosomal aneuploidies. hese include whole-genome sequencing, which is also called massively parallel or shotgun sequencing; chromosome selective or targeted sequencing; and analysis of SNPs (American College of Obstetricians and Gynecologists, 2016a,b). By simultaneously sequencing millions of DNA fragments, investigators can identiy whether the proportion or ratio of fragments from one chromosome is higher than expected. Sequences of fetal DNA are specific to individual chromosomes. hus, samples from women with a Down syndrome fetus have a larger proportion of DNA sequences from chromosome 21. The screening performance of cell-free DNA is excellent. In a metaanalysis of 37 studies of largely high-risk pregnancies, the pooled sensitivity to detect Down syndrome was 99 percent, and to identiy trisomies 18 and 13, 96 and 91 percent, respectively. For each, the speciicity was 99.9 percent (Gil, 2015). The false-positive rate is cumulative for each aneuploidy for which screening is performed, but it is usually below 1 percent. As a result, cell-free DNA screening is recommended as !DNA isolation Cell-free fetal DNA determination Massively parallel genomic sequencing Trisomy 21, 18, and 13screening FIGURE 13-14 Cell-free DNA is actually derived from apoptotic trophoblast. The DNA is isolated from maternal plasma, and real-time quantitative polymerase chain reaction (PCR) may be used to target specific regions or sequences. This may be used for Rh D genotyping, detection of paternally inherited single-gene disorders, or fetal sex determination. Screening for autosomal trisomies and sex chromosomal aneuploidies is performed using whole-genome sequencing, chromosome selective or targeted sequencing, and analysis of single nucleotide polymorphisms.