Tong Yu

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

CONTEXT: Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition that arises from mutations in CYP21A2 gene, which encodes for the steroidogenic enzyme 21-hydroxylase. To prevent genital ambiguity in affected female fetuses, prenatal treatment with dexamethasone must begin on or before gestational week 9. Currently used chorionic villus sampling and amniocentesis provide genetic results at approximately 14 weeks of gestation at the earliest. This means that mothers who want to undergo prenatal dexamethasone treatment will be unnecessarily treating seven of eight fetuses (males and three of four unaffected females), emphasizing the desirability of earlier genetic diagnosis in utero. OBJECTIVE: The objective of the study was to develop a noninvasive method for early prenatal diagnosis of fetuses at risk for CAH. PATIENTS: Fourteen families, each with a proband affected by phenotypically classical CAH, were recruited. DESIGN: Cell-free fetal DNA was obtained from 3.6 mL of maternal plasma. Using hybridization probes designed to capture a 6-Mb region flanking CYP21A2, targeted massively parallel sequencing (MPS) was performed to analyze genomic DNA samples from parents and proband to determine parental haplotypes. Plasma DNA from pregnant mothers also underwent targeted MPS to deduce fetal inheritance of parental haplotypes. RESULTS: In all 14 families, the fetal CAH status was correctly deduced by targeted MPS of DNA in maternal plasma, as early as 5 weeks 6 days of gestation. CONCLUSIONS: MPS on 3.6 mL plasma from pregnant mothers could potentially provide the diagnosis of CAH, noninvasively, before the ninth week of gestation. Only affected female fetuses will thus be treated. Our strategy represents a generic approach for noninvasive prenatal testing for an array of autosomal recessive disorders.

Abstract Real‐time monitoring of wound healing remains a major challenge in clinical tissue regeneration, calling the need for the development of biomaterial‐guided on‐site monitoring wound healing technology. In this study, multifunctional double colorimetry‐integrated polyacrylamide‐quaternary ammonium chitosan‐carbon quantum dots (CQDs)‐phenol red hydrogels are presented, aiming to simultaneously detect the wound pH level, reduce bacterial infection, and promote wound healing. The hybridization of CQDs and pH indicator (phenol red) with the hydrogels enables their high responsiveness, reversibility, and accurate indication of pH variability to reflect the dynamic wound status in the context of both ultraviolet and visible light. Furthermore, these visual images can be collected by smartphones and converted into on‐site wound pH signals, allowing for a real‐time evaluation of the wound dynamic conditions in a remote approach. Notably, the hydrogels exhibit excellent hemostatic and adhesive properties, maintain sufficient wound moisture, and promote wound healing via their high antibacterial activity (against Staphylococcus Aureus , and Escherichia Coli ) and skin repair function. Overall, the resulting hydrogels have high potential as a novel smart and flexible wound dressing platform for theranostic skin regeneration.

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.