The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

Jagged Ends of Urinary Cell-Free DNA: Characterization and Feasibility Assessment in Bladder Cancer Detection.

BACKGROUND: Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. METHODS: Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. RESULTS: The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. CONCLUSIONS: Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing.

OBJECTIVE: This study aimed to provide an individualized assessment of fetal trisomy 21 and trisomy 18 status for twin pregnancies by maternal plasma DNA sequencing. METHOD: Massively parallel sequencing was performed on the plasma/serum DNA libraries of eight twin pregnancies and 11 singleton pregnancies. The apparent fractional fetal DNA concentrations between genomic regions were assessed to determine the zygosities of the twin pregnancies and to calculate the fetal DNA concentrations of each individual member of dizygotic twin pairs. Z-scores were determined for the detection of trisomy 18 and trisomy 21. RESULTS: Circulating DNA sequencing showed elevated chromosome 21 representation in one set of twins and elevated chromosome 18 representation in another pair of twins. Apparent fractional fetal DNA concentration analysis revealed both sets of twins to be dizygotic. The fractional fetal DNA concentrations for each individual fetus of the dizygotic twin pregnancies were determined. Incorporating the information about the fetal DNA fraction, we ascertained that each fetus contributed adequate amounts of DNA into the maternal circulation for the aneuploidy test result to be interpreted with confidence. CONCLUSION: Noninvasive prenatal assessment of fetal chromosomal aneuploidy for twin pregnancies can be achieved with the use of massively parallel sequencing of cell-free DNA in maternal blood.

Prenatal assessment of fetal chromosomal and genetic disorders through maternal plasma DNA analysis.

The existence of cell free DNA derived from the fetus in the plasma of pregnant women was first demonstrated in 1997. This discovery offered the possibility of non-invasive sampling of fetal genetic material simply through the collection of a maternal blood sample. Such cell free fetal DNA molecules in the maternal circulation have subsequently been shown to originate from the placenta and could be detected from about 7 weeks of gestation. It has been shown that cell free fetal DNA analysis could offer highly accurate assessment of fetal genotype and chromosomal makeup for some applications. Thus, cell free fetal DNA analysis has been incorporated as a part of prenatal screening programs for the prenatal management of sex-linked and sex-associated diseases, rhesus D incompatibility as well as the prenatal detection of Down's syndrome.Cell free fetal DNA analysis may lead to a change in the way prenatal assessments are made.

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.

OBJECTIVES: To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. DESIGN: Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. SETTING: Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. PARTICIPANTS: 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. MAIN OUTCOME MEASURES: Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. RESULTS: Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. CONCLUSION: Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

A strategy for identifying circulating placental RNA markers for fetal growth assessment.

OBJECTIVE: To evaluate whether circulating placental mRNAs in maternal plasma could serve as markers for the assessment of fetal growth or intrauterine growth restriction (IUGR). METHODS: From a panel of placental transcripts detectable in maternal plasma identified by microarray previously, we chose growth-related transcripts, namely CSH1, GH2, KISS1, and ADAM12, as potential growth markers. Relationships between the maternal plasma mRNA concentrations with several fetal growth indicators were studied. Maternal plasma mRNA concentrations from IUGR pregnancies with or without pre-eclampsia (PET) were compared with gestational age matched controls cross-sectionally and longitudinally. The four transcripts were quantified by one-step real-time RT-PCR. RESULTS: Maternal plasma GH2 mRNA significantly correlated with birth weight and fetal biometric measurements. Maternal plasma ADAM12 mRNA concentration was significantly higher in IUGR with PET than normal pregnancies in the cross-sectional comparison. No significant difference was observed for all markers between IUGR without PET and controls in both the cross-sectional and longitudinal comparisons. CONCLUSION: This study presents a potential strategy in identifying surrogate markers for the study of fetal growth. Circulating GH2 mRNA in maternal plasma appeared to be associated with fetal growth. The utility of this strategy and the currently assessed markers could be explored in further studies.