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Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma

Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 01/2009; 105(51):20458-63. DOI: 10.1073/pnas.0810641105
Source: PubMed

ABSTRACT Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

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    ABSTRACT: The aim of this study is to determine the fetus Y-STR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and known paternity. All participants signed informed consent and the local ethics committee approved the study. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, the first consecutive 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The median gestational age was 12 weeks (range 12-36). All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses’ haplotypes (Yfiler format) were compared to other 5,328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS 458 locus due to the loss of one repeat unit and 3 cases showed one DYS 385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses’ haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively. Three fetuses’ haplotypes occurred twice in YHRD, which corresponded to paternity probability of 99.9437%. In conclusion, high discriminatory fetal Y-STR haplotype could be determined from maternal plasma during pregnancy starting at 12 weeks of gestation. All male fetuses could be attributed to the alleged father male lineage early in pregnancy. The high probability of paternity associated with each case suggests that the relationship is not random and this strategy can be use as an alternative for male fetal kinship analysis.
    Forensic Science International: Genetics 11/2014; 15. DOI:10.1016/j.fsigen.2014.11.006 · 3.20 Impact Factor
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    • "The motivation of our work is to compare between different bias correction protocols in order to recommend an optimal correction procedure . Since optimization of the test statistic can be considered a separate research area , we use the simple Z - score method described by Chiu et al . in 2008 and 2011 [ 7 , 19 ] , to benchmark the results of our correction methods . Furthermore , the Z - score method has been the basis of many of the subsequent tests so we used this test statistic for our comparisons . "
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    ABSTRACT: Pregnant women carry a mixture of cell-free DNA fragments from self and fetus (non-self) in their circulation. In recent years multiple independent studies have demonstrated the ability to detect fetal trisomies such as trisomy 21, the cause of Down syndrome, by Next-Generation Sequencing of maternal plasma. The current clinical tests based on this approach show very high sensitivity and specificity, although as yet they have not become the standard diagnostic test. Here we describe improvements to the analysis of the sequencing data by reducing GC bias and better handling of the genomic repeats. We show substantial improvements in the sensitivity of the standard trisomy 21 statistical tests, which we measure by artificially reducing read coverage. We also explore the bias stemming from the natural cleavage of plasma DNA by examining DNA motifs and position specific base distributions. We propose a model to correct this fragmentation bias and observe that incorporating this bias does not lead to any further improvements in the detection of fetal trisomy. The improved bias corrections that we demonstrate in this work can be readily adopted into existing fetal trisomy detection protocols and should also lead to improvements in sub-chromosomal copy number variation detection.
    PLoS ONE 01/2014; 9(1):e86993. DOI:10.1371/journal.pone.0086993 · 3.53 Impact Factor
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    • "Currently, two methods have been developed and validated, and have demonstrated a near 100% accuracy. One method is based on next generation sequencing (Fan et al., 2008; Chiu et al., 2008), and the other one is based on MeDIP and real time qPCR (Papageorgiou et al., 2009, 2011). NIPD by next generation sequencing is achieved by high throughput shotgun sequencing of DNA from plasma of maternal peripheral blood, followed by ratio analysis of each chromosome sequence tag density over the median tag density of all autosomes using a z-score analysis (Fan et al., 2008). "
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