Dnase1l3 deletion causes aberrations in length and end-motif frequencies in plasma DNA

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3−/− mice carrying Dnase1l3+/− fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3. Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity.

High-Resolution Profiling of Fetal DNA Clearance from Maternal Plasma by Massively Parallel Sequencing

BACKGROUND With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in maternal plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS Using paired-end MPS, we analyzed serial samples of maternal plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS MPS of maternal plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

Aneuploidy Detection in Mixed DNA Samples by Methylation-Sensitive Amplification and Microarray Analysis

Background: Cell-free fetal nucleic acid, believed to be derived from the placenta/trophoblast, is present in the plasma of pregnant women; however, its use for predictive genetic testing has been severely limited because the circulating fetal DNA is present in a small quantity and mixed with a much larger quantity of maternal DNA. Methods for detecting fetal aneuploidy from the cell-free fetal DNA in plasma are highly sought after, but proposed methods must take into account the small quantity and highly contaminated nature of the available fetal DNA. Methods: We developed a method for methylation-sensitive amplification of DNA suitable for use with small (approximately 1 ng) samples. We used this method in conjunction with 2-color microarray analysis with a custom-made array to investigate whether relative amplification, and hence relative methylation, could be evaluated for a large number of genomic loci. Results: Microarray assessment of genomic methylation accurately predicted the degree of methylation measured with bisulfite-conversion PCR and confirmed that DNA from first-trimester trophoblast was generally hypomethylated compared with whole-blood DNA. With a series of 3 samples in which 1 ng of DNA from a trisomic first trimester placenta was mixed with 9 ng of chromosomally normal peripheral blood DNA, we observed that the microarray signal associated with the trisomic chromosome was significantly different from that of the other chromosomes (P < 0.001). Conclusions: This method has potential to be used for noninvasive detection of fetal aneuploidy.