Prenatal Screening for 22q11.2 Deletion Using a Targeted Microarray-Based Cell-Free DNA Test.

OBJECTIVE: To determine the performance of a targeted microarray-based cell-free DNA (cfDNA) test (Harmony Prenatal Test®) for the identification of pregnancies at increased risk for 22q11.2 deletion. METHODS: Test performance was determined in 2 steps including a total of 1,953 plasma samples. Analytical validation was performed in 1,736 plasma samples. Clinical verification of performance was performed in an additional 217 prospectively ascertained samples from pregnancies with fetal deletion status determined by diagnostic testing. RESULTS: Analytical sensitivity was 75.4% (95% CI: 67.1-82.2%) based on 122 samples with deletions ranging from 1.96 to 3.25 Mb. In 1,614 presumed unaffected samples, specificity was determined to be at least 99.5% (95% CI: 99.0-99.7%). In the clinical cohort, 5 of 7 samples from pregnancies affected with 22q11.2 deletion were determined to have a high probability of deletion. There were no false positive results in the 210 unaffected samples in this cohort. These clinical data are consistent with the performance demonstrated in the analytical validation. CONCLUSIONS: cfDNA testing using a targeted microarray-based technology is able to identify pregnancies at increased risk for 22q11.2 deletions of 3.0 Mb and smaller while maintaining a low false positive rate.

Identifying Robertsonian Translocation Carriers by Microarray-Based DNA Analysis.

OBJECTIVE: To develop a noninvasive prenatal testing improvement that allows identification of Robertsonian translocation carriers. METHODS: Blood samples from 191 subjects, including 7 pregnant and 9 non-pregnant Robertsonian translocation carriers, were analyzed for fetal trisomy and Robertsonian translocation status. Digital Analysis of Selected Regions (DANSR™) assays targeting sequences common to the p arms of 5 acrocentric chromosomes were developed and added to existing DANSR assays. DANSR products were hybridized onto a custom DNA microarray for DNA analysis. The Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm measures the fraction of fetal DNA and accounts for both the fetal and maternal fractions in the cell-free DNA sample to determine Robertsonian risk. The expectation in a Robertsonian translocation carrier is that DANSR assays on acrocentric p arms should have a concentration 20% less than that of controls. RESULTS: The FORTE algorithm correctly classified the fetal trisomy status and maternal Robertsonian translocation status in all 191 samples. Sixteen samples had a Robertsonian risk score above 99%, while 175 samples had a Robertsonian risk score below 0.01%. CONCLUSIONS: Robertsonian translocations are the most common chromosomal translocations and can have significant reproductive consequences. A maternal screen for Robertsonian translocation carriers would provide women valuable information regarding the risk of fetal trisomy.

Mesenchymal cells. Defining a mesenchymal progenitor niche at single-cell resolution.

Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis.

Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineage-tracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardial-derived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.

Microarray-based cell-free DNA analysis improves noninvasive prenatal testing.

OBJECTIVE: To develop a microarray-based method for noninvasive prenatal testing (NIPT) and compare it with next-generation sequencing. METHODS: Maternal plasma from 878 pregnant women, including 187 trisomy cases (18 trisomy 13, 37 trisomy 18, 132 trisomy 21), was evaluated for trisomy risk. Targeted chromosomes were analyzed using Digital Analysis of Selected Regions (DANSR™) assays. DANSR products were subsequently divided between two DNA quantification methods: microarrays and next-generation sequencing. For both microarray and sequencing methodologies, the Fetal-Fraction Optimized Risk of Trisomy Evaluation (FORTE™) algorithm was used to determine trisomy risk, assay variability across samples, and compute fetal fraction variability within samples. RESULTS: NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing. The assay variability, a measure of variance of chromosomal cfDNA counts, was lower for microarrays than for sequencing, 0.051 versus 0.099 (p < 0.0001). Analysis time using microarrays was faster, 7.5 versus 56 h for sequencing. Additionally, fetal fraction precision was improved 1.6-fold by assaying more polymorphic sites with microarrays (p < 0.0001). Microarrays correctly classified all trisomy and nontrisomy cases. CONCLUSIONS: NIPT using microarrays delivers more accurate cfDNA analysis than next-generation sequencing and can be performed in less time.