Noninvasive prenatal detection of fetal subchromosomal copy number aberrations (CNAs) can be achieved through massively parallel sequencing of maternal plasma DNA. However, when a mother herself is a ...carrier of a CNA, one cannot discern if her fetus has inherited the CNA. In addition, false-positive results would become more prevalent when more subchromosomal regions are analyzed.
We used a strategy that combined count- and size-based analyses of maternal plasma DNA for the detection of fetal subchromosomal CNAs in 7 target regions for 10 test cases.
For the 5 cases in which CNAs were present only in the fetus, the size-based approach confirmed the aberrations detected by the count-based approach. For the 5 cases in which the mother herself carried an aberration, we successfully deduced that 3 of the fetuses had inherited the aberrations and that the other 2 fetuses had not inherited the aberrations. No false positives were observed in this cohort.
Combined count- and size-based analysis of maternal plasma DNA permits the noninvasive elucidation of whether a fetus has inherited a CNA from its mother who herself is a carrier of the CNA. This strategy has the potential to improve the diagnostic specificity of noninvasive prenatal testing.
Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. ...We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.
•Genomewide methylation signatures can infer the tissue of origin of urinary cfDNA.•Large variation in the proportional contribution of cfDNA from different tissues•Urinary cfDNA is fragmented under first-order kinetics in a time dependent manner.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA ...molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's
= 0.71 vs. -0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in
-deficient mice but higher in
-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.
Objective
Long cell‐free DNA (cfDNA) can be found in the plasma of pregnant women and cancer patients. We investigated if droplet digital PCR (ddPCR) can analyze such molecules for diagnostic ...purposes using preeclampsia as a model.
Method
Plasma samples from ten preeclamptic and sixteen normal pregnancies were analyzed. Two ddPCR assays targeting a single‐copy gene, VCP, and one ddPCR assay targeting LINE‐1 repetitive regions were used to measure the percentages of long cfDNA >533, 1001, and 170 bp, respectively. The LINE‐1 assay was developed as guided by in silico PCR analyses to better differentiate preeclamptic and normal pregnancies.
Results
Preeclamptic patients had a significantly lower median percentage of long cfDNA than healthy pregnant controls, as determined by the LINE‐1 170 bp assay (28.9% vs. 35.1%, p < 0.0001) and the VCP 533 bp assay (6.6% vs. 8.7%, p = 0.014). The LINE‐1 assay provided a better differentiation than the VCP 533 bp assay (area under ROC curves, 0.94 vs. 0.79).
Conclusion
ddPCR is a cost‐effective approach for unlocking diagnostic information carried by long cfDNA in plasma and may have applications for the detection of preeclampsia. Further longitudinal studies with larger cohorts are required to assess the clinical utility of this test.
Key points
What's already known about this topic?
Using long‐read sequencing, a significant percentage of long cfDNA was detected in the plasma of pregnant women. The percentages of long cfDNA >500 bp and >1000 bp were 32.3% and 22.0%, respectively, as determined by single molecule real‐time (SMRT) sequencing, and 5.4% and 0.79%, respectively, as determined by nanopore sequencing.
A reduction in the percentage of long cfDNA was found in plasma from preeclamptic patients by SMRT sequencing.
What does this study add?
Using droplet digital PCR (ddPCR), the percentages of long cfDNA >533 bp and >1001 bp in the plasma of normal pregnancies were 8.7% and 4.6%, respectively.
Size analysis of cfDNA in maternal plasma using the ddPCR assay targeting LINE‐1 regions is a new type of biomarker for preeclampsia.
ddPCR is a cost‐effective method for unlocking the diagnostic information of long cfDNA in plasma.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract Background The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust ...methods for targeted haplotyping. Methods We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). Results For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. Conclusions The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.
Nuclear-derived cell-free DNA (cfDNA) molecules in blood plasma are nonrandomly fragmented, bearing a wealth of information related to tissues of origin. DNASE1L3 (deoxyribonuclease 1 like 3) is an ...important player in shaping the fragmentation of nuclear-derived cfDNA molecules, preferentially generating molecules with 5 CC dinucleotide termini (i.e., 5 CC-end motif). However, the fragment end properties of microbial cfDNA and its clinical implication remain to be explored.
We performed end motif analysis on microbial cfDNA fragments in plasma samples from patients with sepsis. A sequence context-based normalization method was used to minimize the potential biases for end motif analysis.
The end motif profiles of microbial cfDNA appeared to resemble that of nuclear cfDNA (Spearman correlation coefficient: 0.82, P value 0.001). The CC-end motif was the most preferred end motif in microbial cfDNA, suggesting that DNASE1L3 might also play a role in the fragmentation of microbe-derived cfDNA in plasma. Of note, differential end motifs were present between microbial cfDNA originating from infection-causing pathogens (enriched at the CC-end) and contaminating microbial DNA potentially derived from reagents or the environment (nearly random). The use of fragment end signatures allowed differentiation between confirmed pathogens and contaminating microbes, with an area under the receiver operating characteristic curve of 0.99. The performance appeared to be superior to conventional analysis based on microbial cfDNA abundance alone.
The use of fragmentomic features could facilitate the differentiation of underlying contaminating microbes from true pathogens in sepsis. This work demonstrates the potential usefulness of microbial cfDNA fragmentomics in metagenomics analysis.
Liquid biopsy using cell-free DNA (cfDNA) has gained global interest as a molecular diagnostic tool. However, the analysis of cfDNA in cancer patients and pregnant women has been focused on short DNA ...molecules (e.g., ≤ 600 bp). With the detection of long cfDNA in the plasma of pregnant women and cancer patients in two recent studies, a new avenue of long cfDNA-based liquid biopsy has been opened. In this review, we summarize our current knowledge in this nascent field of long cfDNA analysis, focusing on the fragmentomic and epigenetic features of long cfDNA. In particular, long-read sequencing enabled single-molecule methylation analysis and subsequent determination of the tissue-of-origin of long cfDNA, which has promising clinical potential in prenatal and cancer testing. We also examine some of the limitations that may hinder the immediate clinical applications of long cfDNA analysis and the current efforts involved in addressing them. With concerted efforts in this area, it is hoped that long cfDNA analysis will add to the expanding armamentarium of liquid biopsy.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Magnetotactic bacteria have evolved complex subcellular machinery to construct linear chains of magnetite nanocrystals that allow the host cell to sense direction. Each mixed-valent iron nanoparticle ...is mineralized from soluble iron within a membrane-encapsulated vesicle termed the magnetosome, which serves as a specialized compartment that regulates the iron, redox, and pH environment of the growing mineral. To dissect the biological components that control this process, we have carried out a genetic and biochemical study of proteins proposed to function in iron mineralization. In this study, we show that the redox sites of c -type cytochromes of the Magnetospirillum magneticum AMB-1 magnetosome island, MamP and MamT, are essential to their physiological function and that ablation of one or both heme motifs leads to loss of function, suggesting that their ability to carry out redox chemistry in vivo is important. We also develop a method to heterologously express fully heme-loaded MamP from AMB-1 for in vitro biochemical studies, which show that its Fe(III)–Fe(II) redox couple is set at an unusual potential (−89 ± 11 mV) compared with other related cytochromes involved in iron reduction or oxidation. Despite its low reduction potential, it remains competent to oxidize Fe(II) to Fe(III) and mineralize iron to produce mixed-valent iron oxides. Finally, in vitro mineralization experiments suggest that Mms mineral-templating peptides from AMB-1 can modulate the iron redox chemistry of MamP.
Significance Biomineralization is an important and widespread phenomenon by which living systems produce solid materials from soluble metal ions and has key implications with regard to environmental and health processes. In this work, we report studies of redox and structural components that control mineralization events in the production of iron nanoparticles in directional-sensing bacteria.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
To review the safety and efficacy of linear accelerator-based stereotactic radiosurgery (SRS) for brainstem metastases. We reviewed all patients with brain metastases treated with SRS at DF/BWCC from ...2001 to 2009 to identify patients who had SRS to a single brainstem metastasis. Overall survival and freedom-from-local failure rates were calculated from the date of SRS using the Kaplan–Meier method. Prognostic factors were evaluated using the log-rank test and Cox proportional hazards model. A total of 24 consecutive patients with brainstem metastases had SRS. At the time of SRS, 21/24 had metastatic lesions elsewhere within the brain. 23/24 had undergone prior WBRT. Primary diagnoses included eight NSCLC, eight breast cancer, three melanoma, three renal cell carcinoma and two others. Median dose was 13 Gy (range, 8–16). One patient had fractionated SRS 5 Gy ×5. Median target volume was 0.2 cc (range, 0.02–2.39). The median age was 57 years (range, 42–92). Follow-up information was available in 22/24 cases. At the time of analysis, 18/22 patients (82%) had died. The median overall survival time was 5.3 months (range, 0.8–21.1 months). The only prognostic factor that trended toward statistical significance for overall survival was the absence of synchronous brain metastasis at the time of SRS; 1-year overall survival was 31% with versus 67% without synchronous brain metastasis (log rank
P
= 0.11). Non-significant factors included primary tumor histology and status of extracranial disease (progressing vs. stable/absent). Local failure occurred in 4/22 cases (18%). Actuarial freedom from local failure for all cases was 78.6% at 1 year. RTOG grade 3 toxicities were recorded in two patients (ataxia, confusion). Linac-based SRS for small volume brainstem metastases using a median dose of 13 Gy is associated with acceptable local control and low morbidity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of ...protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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