Investigation of biological factors influencing the placental mRNA profile in maternal plasma.

Objective: Circulating placental-derived RNA is useful for noninvasive prenatal investigation. However, in addition to placental gene expression, there are limited investigations on other biological parameters that may affect the circulating placental RNA profile. In this study, we explored two of these potential parameters.

High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing.

Background: Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Synergy of total PLAC4 RNA concentration and measurement of the RNA single-nucleotide polymorphism allelic ratio for the noninvasive prenatal detection of trisomy 21.

Background: Maternal plasma mRNA encoded by the PLAC4 gene (placenta-specific 4), which is transcribed from chromosome 21 in placental cells, is a potential marker for the noninvasive assessment of chromosome 21 dosage in the fetus. We evaluated the diagnostic sensitivities and specificities of 2 trisomy 21-screening approaches that use maternal plasma PLAC4 mRNA.

Methods: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses.

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma.

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved.

Mass spectrometry-based detection of hemoglobin E mutation by allele-specific base extension reaction.

Background: The specific detection of a minor population of mutant DNA molecules requires methods of high specificity and sensitivity. While the single-allele base extension reaction (SABER) was shown to be useful for the detection of certain beta-thalassemia mutations, we encountered problems with false positivity during development of SABER for the noninvasive prenatal diagnosis of the hemoglobin E (HbE) disease. Systematic optimization resulted in an alternative protocol, the allele-specific base extension reaction (ASBER).

Quantitative subtyping of hepatitis B virus reveals complex dynamics of YMDD motif mutants development during long-term lamivudine therapy.

Background/aims: Treatment of chronic hepatitis B (CHB) with lamivudine (3TC) is limited by development of drug-resistant mutants at the YMDD motif. We aimed to validate the use of mass spectrometry to detect YMDD mutants and quantify viral subpopulations.

Methods: A total of 21 Chinese patients with severe acute exacerbation of CHB treated with 3TC were studied.

Reduced plasma RNA integrity in nasopharyngeal carcinoma patients.

Purpose: Recent research has shown the feasibility of detecting cell-free RNA markers in human subjects. As elevated RNase activity has previously been described in the circulation of cancer patients, we hypothesized that cancer patients may have reduced plasma RNA integrity. In this study, we used nasopharyngeal carcinoma (NPC) as a model system to test this hypothesis.

Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients.

Despite the increasing clinical applications of circulating EBV DNA analysis as a tumor marker, the molecular nature of these EBV DNA molecules remains unclear. We subjected plasma/serum samples of nasopharyngeal carcinoma and lymphoma patients to DNase digestion and ultracentrifugation and showed that circulating EBV DNA molecules are "naked" DNA fragments instead of being contained inside virions. We further showed that these EBV DNA fragments were relatively short, and 87% of them were shorter than 181 bp.

Quantitative analysis of pleural fluid cell-free DNA as a tool for the classification of pleural effusions.

Background: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid.

Methods: We recruited 50 consecutive patients with pleural effusions with informed consent.

Prenatal exclusion of beta thalassaemia major by examination of maternal plasma.

The discovery of the presence of fetal DNA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of beta thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (-CTTT) mutation in the beta globin gene from maternal plasma by real-time PCR.

Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma.

Background: Fetal DNA has been detected in maternal plasma by the use of genetic differences between mother and fetus. We explore the possibility of using epigenetic markers for the specific detection of fetal DNA in maternal plasma.

Methods: A differentially methylated region in the human IGF2-H19 locus and a single-nucleotide polymorphism in this region were chosen for the study.