| There are many kinds of complex etiology for birth defects. Down syndrome (DS) is one of them caused by the presence of all or part of a third copy of chromosome 21. DS can lead to intrauterine growth retardation, stillbirth and miscarriage. It is typically associated with moderate intellectual disability, an increased risk of heart, digestive system and other deformities in the survival children. The incidence of DS in the neonatal is 1/600-1/800. In China, the family and social economic burden in every DS patient’s life is about 800,000RMB.The economic burden from the the annual increase in new DS is more than 10 billion RMB.Because there is no effective therapy for Down syndrome, guidelines recommend that screening for Down syndrome be offered to all pregnant women, regardless of age. A number of tests can be used, with varying levels of accuracy. They are usually used in combination to increase the detection rate, while maintaining a low false positive rate. None can be definitive, thus if screening is positive either amniocentesis or chorionic villous sampling is required to confirm the diagnosis. Screening in both the first and second trimesters is better than just screening in the first trimester. The different screening techniques in use are able to pick up 90 to 95% of cases with a false positive rate of between 2 and 5%.With the rapid development of next-generation sequencing technology, the advantage of NIPT with cffDNA in the maternal plasma is paid more and more attention. Now it is one of the hot issues in the research field of prenatal diagnosis and applied in the clinical diagnosis. With its technical and commercial advancement, test sensitivities and specificities for T21, T18 and T13 is close to 99%. The sensitivity and specificity are slightly lower for the sex chromosomes. However, it has to be defined as prenatal screening due to the false negative and false positive results.But as prenatal screening platform, its high cost, long cycle and not suitable for clinical report are embarrassing shortcomings in the field of prenatal screening. Therefore, it is very important to solve the cause for the discrepancy between NIPT and karyotyping results.Our research targeted at the discordance between NIPT and karyotyping results in clinical cases to explore the causes and the solutions. Trisomy 21 false negatives will bring trisomy 21 children which is a serious consequence. So in the first part research, we explore the causes of false negative NIPT result by the detection and analysis of two false negative placental samples, and then we try to find out the solutions for this problem.The second part is based on the background that there are many kinds of sex chromosome karyotype abnormalities and there are huge phenotypic differences in the children. Some phenotypes (such as 47, xxx) have no influence on the quality of life. However the false positive result will always bring panic in the family or leading to unnecessary selective abortion, especially in our country with high standard requirement in birth control. Therefore we explore the causes of false positive result in sex chromosome NIPT detection, and then try to find out the solutions for this problem.Clinical application of existing massively parallel sequencing shows.many shortcomings, such as high cost, long operating cycle and not conducive to widespread. High-throughput semiconductor sequencing technology has been proven better be used for fetal aneuploidies NIPT. In the third part, we conducted a comparative analysis of the performance of four different Ion chips on detecting fetal chromosomal aneuploidies for the option of most suitable ion torrent chips in the clinical application.Part I Influence of Placental Mosaicism on False Negative Associated With NIPT of Down syndromeObjectives We explore the causes of false negative NIPT result by the detection and analysis of two false negative placental samples to provide another explanation for the false negative result in NIPT.Methods The fetal DNA fraction for female fetus was determined from the ratio of methylated RASSFIA fetal DNA to non-methylated maternal RASSFIA DNA, as measured by quantitative polymerase chain reaction. For male fetus, the fetal DNA fraction was determined from the relative fetal contribution of Y chromosome sequences. Eleven genomic DNA samples extracted from white blood cells were used as normal reference controls (six are shown as normal control) and T21 DNA used as a positive control. Test fetal DNA samples were HT, heart tissue; and LT, liver tissue. Placental DNA samples were RUC, root of umbilical cord; MUC, middle of umbilical cord; CP, center of placenta, CMS, center of maternal side; and EMS, edge of maternal side. Chromosome representation (CR) of Ch21 in each sample was calculated by Ch21 sequence reads/total sequence reads as previously described. Levels of T21 mosaicism were subsequently determined using the following formula: CR Ch21 in test sample/mean CR of Ch21 in the reference sample×100%.Results The fetal DNA fraction in the maternal plasma sample was calculated at 15.55% in the first case. DNA sequencing of fetal and placental samples showed that umbilical cord at the midpoint and the placental base had T21 mosaicism levels of 90% and 53%, respectively. In contrast, placental biopsies taken at the center of the tissue and, at the middle and edge of the maternal-fetal interface, had variable T21 mosaicism levels of 23%,17%, and 21%, respectively. Normal placental tissue sampled at similar sites showed no evidence of T21 mosaicism. The fetal DNA fraction in the maternal plasma sample was calculated at 19.72% in the second case.Placental tissue recovered for evidence of placental mosaicism. Fetal liver, heart, and umbilical cord sampled at the midpoint and base of the placenta had T21 mosaicism levels>92%. In contrast, placental biopsies taken at the center of the tissue and at the middle and edge of the maternal-fetal interface had variable T21 mosaicism levels of 76%,51%, and 2.3%, respectively.Conclusions1. The false negative non-invasive prenatal test results reported are believed to be due to a low effective fetal DNA fraction in maternal plasma and/or placental mosaicism.2. The degree and compartmentalization of placental mosaicism can potentially reduce the effective output of fetal DNA into the maternal circulation to steady state levels below the detection limit of non invasive prenatal testing, leading to a false negative result.Part Ⅱ Influence of Maternal Mosaicism on False Positive Associated With NIPT of Sex Chromosomal AneuploidiesObjectives In the human fetus, sex chromosome aneuploidies (SCAs) are as prevalent as the common autosomal trisomies 21,18, and 13. Currently, most noninvasive prenatal tests (NIPTs) offer screening only for chromosomes 21,18, and 13, because the sensitivity and specificity are markedly higher than for the sex chromosomes. Limited studies suggest that the reduced accuracy associated with detecting SCAs is due to confined placental, placental, or true fetal mosaicism.We aimed to demonstrate that an altered maternal karyotype may also be an important contributor to discordant SCA NIPT results.Methods We developed a rapid karyotyping method that uses massively parallel sequencing to measure the degree of chromosome mosaicism. The method was validated with DNA models mimicking XXX and XO mosaicism and then applied to maternal white blood cell (WBC) DNA from patients with discordant SCA NIPT results.Results Sequencing karyotyping detected chromosome X (ChrX) mosaicism as low as 5%, allowing an accurate assignment of the maternal X karyotype. In a prospective NIPT study, we showed that 16 (8.6%) of 187 positive SCAs were due to an abnormal maternal ChrX karyotype that masked the true contribution of the fetal ChrX DNA fraction.Conclusion The accuracy of NIPT for ChrX and ChrY can be improved substantially by integrating the results of maternal-plasma sequencing with those for maternal-WBC sequencing. The relatively high frequency of maternal mosaicism warrants mandatory WBC testing in both shotgun sequencing-and single-nucleotide polymorphism-based clinical NIPT after the finding of a potential fetal SCA.Part III Assessment of Ion Torrent Chips Associated With NIPTObjectives We conducted a comparative analysis of the performance of four different Ion chips on detecting fetal chromosomal aneuploidies for the option of most suitable ion torrent chips in the clinical application.Methods Eight maternal plasma DNA samples, including four pregnancies with normal fetuses and four with trisomy 21 fetuses, were sequenced on Ion Torrent 314/316/318/PI chips respectively. Results such as read mapped ratio, correlation coefficient, and phred quality score were calculated and parallel compared.Results All samples were correctly classified even with low throughput chip, and among the four chips, the 316 chip had the highest read mapped ratio, correlation coefficient, mean read length and phred quality score. All chips were well consistent with each other.Conclusion Our results showed that all Ion chips are applicable in noninvasive prenatal fetal aneuploidy diagnosis. We recommend researchers or clinicians to use appropriate chip with bar coding technology based on the sample number. |
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