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

Abstract

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.

Keywords: cell-free DNA; cfDNA; liquid biopsy; noninvasive prenatal testing; systemic lupus erythematosus.

Fig. 1.

Size distributions of plasma DNA fragments in the range from (A) 0–600 bp on linear scale, (B) 0–600 bp on logarithmic scale, and (C) 20–120 bp on linear scale. The mean size distribution for different groups is shown in different colors. The black, gray, red, blue, and green lines represent the average size profile of plasma DNA molecules originating from 9 WT mice, 3 mice with Dnase1 deletion (Dnase1^−/−), 13 mice with Dnase1l3 deletion (Dnase1l3^−/−), 2 mice with Dnasell3 and Dnase1 double deletion (Dnasell3^−/− & Dnase1^−/−), and 5 mice with Dnase1l3 and Cd40lg double deletion (Dnasell3^−/− & Cd40lg^−/−), respectively.

Fig. 2.

Proportion of long plasma DNA fragments among groups. The proportion of long DNA fragments was determined from the percentage of sequenced reads with a size greater than 250 bp. Samples from 9 WT mice, 3 Dnase1^−/− mice, 13 Dnase1l3^−/− mice, 2 Dnase1^−/−/Dnase1l3^−/− mice, and 5 Dnasell3^−/−/Cd40lg^−/− mice were sequenced. Each circle represents one mouse. Statistical difference in the percentage between WT mice and mice with Dnase1l3 deletion was calculated by using the Wilcoxon rank-sum test.

Fig. 3.

Percentages of plasma DNA fragments with the selected motifs among groups. The select motifs CCCA, CCTG, CCAG, CCAA, CCAT, and CCTC are the top six 4-mer motifs ranked with the highest frequencies in the WT group and show a statistically significant reduction in Dnase1l3^−/− mice. Samples from 9 WT mice, 3 Dnase1^−/− mice, 13 Dnase1l3^−/− mice, 2 Dnase1^−/−/Dnase1l3^−/− mice, and 5 Dnasell3^−/−/Cd40lg^−/− mice were sequenced. Each circle represents one mouse. Statistical difference in the percentage between WT mice and mice with Dnase1l3 deletion was calculated by using the Wilcoxon rank-sum test.

Fig. 4.

Mean size distributions of plasma DNA fragments in pregnant mice. Solid black line represents data from two pregnant WT mice carrying WT fetuses. Solid blue line represents data from four pregnant Dnase1l3^−/− mice carrying Dnase1l3^+/− fetuses. Solid red line represents data from three pregnant Dnase1l3^−/− mice carrying Dnase1l3^−/− fetuses.

Fig. 5.

Difference between maternal and fetal DNA size profiles. (A) Size distributions, (B) plot of cumulative size frequencies for maternal DNA (black) and fetal DNA (red), and (C) the difference in cumulative frequencies, denoted as ΔS, between maternal and fetal DNA for a representative WT pregnant mouse (Mu31). (D) Size distributions, (E) plot of cumulative frequencies of plasma DNA size for maternal DNA (black) and fetal DNA (red), and (F) the difference in cumulative frequencies, denoted as ΔS, between maternal and fetal DNA for a representative Dnase1l3^−/− pregnant mouse (Mu47).

Fig. 6.

Percentages of fragments with the six selected motifs, including CCCA, CCTG, CCAG, CCAA, CCAT, and CCTC, in plasma DNA of nonpregnant mice; maternal and fetal DNA fractions in plasma of pregnant mice with different Dnase1l3 genotypes. Each circle represents the data from one mouse. The genotypes of the nonpregnant mice as well as the fetal and maternal DNA subset of pregnant mice are annotated under the x axis. The first two groups are nonpregnant WT and Dnase1l3^−/− mice, respectively. For pregnant mice, the strains of the maternal and fetal DNA subset in plasma are also annotated. B6 and BALB/c are two different mouse strains with different genetic backgrounds. As the motifs of fetal DNA sequences would theoretically affect the measurement of motifs in the shared sequences, the abundance of a motif from maternally derived sequences was adjusted by the fetal DNA fraction as described in Materials and Methods. The adjusted percentages of the selected motifs of the maternal DNA subset are labeled with “(adjusted)“ on the x axis. Statistical difference in the percentage between maternal and fetal DNA subsets of pregnant mice with Dnase1l3 deletion was calculated by using the Wilcoxon rank-sum test.