No abstract

ObjectiveThe fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.MethodsArtificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.ResultsA method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.ConclusionA relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.

SUMMARY: Long noncoding RNAs (lncRNAs) are an important class of pervasive genes, and their misregulation has been shown in various types of diseases. However, the relationship between lncRNAs and the immune response to pathogen infection has been rarely reported. Helicobacter pylori is a major human pathogenic bacterium that causes gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The regulatory mechanism of the H. pylori-induced immune response is not yet clear. In the present study, we identified nonoverlapping signatures of a small number of lncRNAs that were aberrantly expressed in H. pylori-infected gastric epithelial cells using microarray analysis followed by bioassays. From microarray data, we found that 23 lncRNAs were upregulated and 21 were downregulated. Five lncRNAs, XLOC_004562, XLOC_005912, XLOC_000620, XLOC_004122, and XLOC_014388, were further evaluated using quantitative reverse transcription-PCR, and the results matched well with microarray data. In addition, XLOC_004122 and XLOC_ 014388 were decreased in gastric mucosal tissues of H. pylori-positive patients. Differentially expressed lncRNAs may play a partial or key role in the immune response to H. pylori, and this may provide potential targets for the future treatment of H. pylori-related diseases.

Massively parallel sequencing of circulating fetal DNA in the plasma of pregnant women is a common method for noninvasive prenatal testing (NIPT) of fetal trisomy 13, 18, and 21. However, circulating DNA is not restricted to pregnant women, with increased levels of plasma DNA also frequently detected in the plasma of cancer patients. Among pregnant women whose NIPT results were inconsistent with the fetal karyotype, a small number of patients have subsequently been diagnosed with a previously undetected malignancy. However, the extent to which circulating tumor DNA (ctDNA) affects the results of NIPT is still unclear. We examined serum from 50 nonpregnant women with breast tumors by NIPT. These samples were then added to serum containing trisomy 13, 18, and 21 fetal DNA to figure out the extent to which maternal tumors can interrupt NIPT results in pregnant women with breast tumors. Concentrations of cell-free DNA (cfDNA) were higher in both pregnant women and breast tumor patients, relative to nonpregnant healthy controls. Among the 50 samples evaluated, 3 produced false positive NIPT results for trisomy 13, 18, or 21, indicating that genomic copy number variations (CNVs) had occurred. Simulation testing also showed that ctDNA can increase the standard deviation of the associated z-scores, which lower absolute z-scores by decreasing the proportion of circulating fetal DNA relative to total DNA. Of the 50 samples tested, 9 fell within the equivocal range and 8 produced false negative results for trisomy 13, 18, or 21. Data presented here show for the first time that ctDNA is able to affect NIPT results in two ways. First, ctDNA can lead to false positive results due to the detection of genomic CNVs in tumor DNA. Alternatively, ctDNA can increase the likelihood of a false negative by decreasing the proportion of circulating fetal DNA in serum.