cfDNA screening and diagnosis of monogenic disorders - where are we heading?

Cell-free fetal DNA analysis for non-invasive prenatal screening of fetal chromosomal aneuploidy has been widely adopted for clinical use. Fetal monogenic diseases have also been shown to be amenable to non-invasive detection by maternal plasma DNA analysis. A number of recent technological developments in this area has increased the level of clinical interest, particularly as one approach does not require customized reagents per mutation. The mutational status of the fetus can be assessed by determining which parental haplotype that fetus has inherited based on the detection of haplotype-associated SNP alleles in maternal plasma. Such relative haplotype dosage analysis requires the input of the parental haplotype information for interpretation of the fetal inheritance pattern from the maternal plasma DNA data. The parental haplotype information can be obtained by direct means, reducing the need to infer haplotypes using DNA from other family members. The technique also allows the assessment of complex mutations and has multiplexing capabilities where a number of genes and mutations can be assessed at the same time. These advantages allow non-invasive prenatal diagnosis of fetal monogenic diseases to be much more scalable. These applications may drive the next wave of clinical adoption of cell-free fetal DNA testing. © 2018 The Authors Prenatal Diagnosis Published by John Wiley & Sons Ltd.

Genomic analysis of fetal nucleic acids in maternal blood.

The 15 years since the discovery of fetal DNA in maternal plasma have witnessed remarkable developments in noninvasive prenatal diagnosis. An understanding of biological parameters governing this phenomenon, such as the concentration and molecular size of circulating fetal DNA, has guided its diagnostic applications. Early efforts focused on the detection of paternally inherited sequences, which were absent in the maternal genome, in maternal plasma. Recent developments in precise measurement technologies such as digital polymerase chain reaction (PCR) have allowed the detection of minute allelic imbalances in plasma and have catalyzed analysis of single-gene disorders such as the hemoglobinopathies and hemophilia. The advent of massively parallel sequencing has enabled the robust detection of fetal trisomies in maternal plasma. Recent proof-of-concept studies have detected a chromosomal translocation and a microdeletion and have deduced a genome-wide genetic map of a fetus from maternal plasma. Understanding the ethical, legal, and social aspects in light of such rapid developments is thus a priority for future research.

Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research.

The collection of fetal genetic materials is required for the prenatal diagnosis of fetal genetic diseases. The conventional methods for sampling fetal genetic materials, such as amniocentesis and chorionic villus sampling, are invasive in nature and are associated with a risk of fetal miscarriage. For decades, scientists had been pursuing studies with goals to develop non-invasive methods for prenatal diagnosis. In 1997, the existence of fetal derived cell-free DNA molecules in plasma of pregnant women was first demonstrated. This finding provided a new source of fetal genetic material that could be obtained safely through the collection of a maternal blood sample and provided a new avenue for the development of non-invasive prenatal diagnostic tests. Now 15 years later, the diagnostic potential of circulating fetal DNA analysis has been realized. Fruitful research efforts have resulted in the clinical implementation of a number of non-invasive prenatal tests based on maternal plasma DNA analysis and included tests for fetal sex assessment, fetal rhesus D blood group genotyping and fetal chromosomal aneuploidy detection. Most recently, research groups have succeeded in decoding the entire fetal genome from maternal plasma DNA analysis which paved the way for the achievement of non-invasive prenatal diagnosis of many single gene diseases. A paradigm shift in the practice of prenatal diagnosis has begun.

The potential clinical utility of serial plasma albumin mRNA monitoring for the post-liver transplantation management.

OBJECTIVES: Elevated albumin (ALB) mRNA concentration has been reported in the plasma of patients with liver diseases. The plasma ALB mRNA measurement was shown to be an effective indicator of liver pathologies with superior diagnostic sensitivity and specificity when compared with alanine transaminase (ALT). We hypothesized that serial plasma ALB mRNA analysis would be helpful in the early detection and monitoring of post-liver transplantation complications. DESIGN AND METHODS: One hundred and five blood specimens were collected from 24 post-transplant recipients. Biochemical liver function test profiles and plasma ALB mRNA concentrations were assessed. RESULTS: Over the study period, the health status of 14 recipients (58%) remained stable (Stable group). Their plasma ALB mRNA concentrations remained within a low-concentration range. In contrast, 10 recipients (42%) developed 14 episodes of hepatic complications (Unstable group). The median plasma ALB mRNA concentration of the Unstable group was 6.5-times higher than that of the Stable group. Plasma ALB mRNA concentration was elevated on 13/14 (93%) episodes of the hepatic complications while ALT was elevated only on 8/14 (57%) episodes. CONCLUSIONS: The elevation of plasma ALB mRNA may allow sensitive detection of hepatic complications and monitoring of the clinical course in a dynamic fashion. Serial plasma ALB mRNA measurement is potentially useful for post-liver transplantation management.

Noninvasive fetal trisomy 21 detection using chromosome-selective sequencing: a variation of the molecular counting theme.

Fetal DNA is present in the plasma of pregnant women. A fetus with trisomy of a chromosome will release an increased amount of DNA from that chromosome into maternal plasma. Such an increase has previously been measured using methods that allow individual DNA molecules to be counted. One such method involves the use of random massively parallel sequencing of maternal plasma DNA. As the sequencing process is random, sequence tags from a potentially aneuploid chromosome only represent a fraction of the sequencing data. The performance of selective amplification and sequencing of specific genomic regions is a recently reported approach for focusing the sequencing power onto genomic regions of diagnostic interest. This article provides a critical analysis of this approach and puts this method in the perspective of other recent works in the field.

Host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants.

Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.