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: 9.67). 01/2009; 105(51):20458-63. DOI: 10.1073/pnas.0810641105
Source: PubMed


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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    • "-invasive fetal genetic analysis are available at clinical services, they include detection of fetal sex [4], rhesus D blood type [5], fetal aneuploidy [6], paternal-derived mutations [7] and, also, paternity [8]. The cfDNA originates from the placenta cells and apoptosis appears to be the main mechanisms controlling its releases to the mother circulation [9]. "
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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 · 4.60 Impact Factor
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    • "The presence of cell-free DNA released by the fetus into the circulation of its mother was reported in 1997.6 By analyzing this source of fetal genetic material, obtainable through a blood sample from a pregnant woman, gNIPT has been developed7 and proposed as potentially changing the approach to prenatal diagnosis for DS and other conditions. Cell-free fetal DNA (cffDNA) discovered in maternal plasma6 originates from placental cell turnover.8 "
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    ABSTRACT: Current prenatal diagnosis for fetal aneuploidies (including trisomy 21 [T21]) generally relies on an initial biochemical serum-based noninvasive prenatal testing (NIPT) after which women who are deemed to be at high risk are offered an invasive confirmatory test (amniocentesis or chorionic villi sampling for a fetal karyotype), which is associated with a risk of fetal miscarriage. Recently, genomics-based NIPT (gNIPT) was proposed for the analysis of fetal genomic DNA circulating in maternal blood. The diffusion of this technology in routine prenatal care could be a major breakthrough in prenatal diagnosis, since initial research studies suggest that this novel approach could be very effective and could reduce substantially the number of invasive procedures. However, the limitations of gNIPT may be underappreciated. In this review, we examine currently published literature on gNIPT to highlight advantages and limitations. At this time, the performance of gNIPT is relatively well-documented only in high-risk pregnancies for T21 and trisomy 18. This additional screening test may be an option for women classified as high-risk of aneuploidy who wish to avoid invasive diagnostic tests, but it is crucial that providers carefully counsel patients about the test's advantages and limitations. The gNIPT is currently not recommended as a first-tier prenatal screening test for T21. Since gNIPT is not considered as a diagnostic test, a positive gNIPT result should always be confirmed by an invasive test, such as amniocentesis or chorionic villus sampling. Validation studies are needed to optimally introduce this technology into the existing routine workflow of prenatal care.
    The Application of Clinical Genetics 07/2014; 7:127-31. DOI:10.2147/TACG.S35602
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    • "Since the first demonstration of the use of cell-free DNA (cfDNA) to detect aneuploidy (Chiu et al., 2008; Fan et al., 2008), many groups around the world have reported large studies showing high sensitivities and specificities for the detection of trisomies 13, 18 and 21 (Jiang et al., 2012; Palomaki et al., 2011; Sehnert et al., 2011). In contrast to invasive methods such as amniocentesis, non-invasive prenatal testing (NIPT) uses cfDNA in maternal plasma and does not present a risk of miscarriage. "
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    ABSTRACT: Non-invasive prenatal testing (NIPT) of fetal aneuploidy using cell-free fetal DNA is becoming part of routine clinical practice. RAPIDR (Reliable Accurate Prenatal non-Invasive Diagnosis R package) is an easy-to-use open-source R package that implements several published NIPT analysis methods. The input to RAPIDR is a set of sequence alignment files in the BAM format, and the outputs are calls for aneuploidy, including trisomies 13, 18, 21 and monosomy X as well as fetal sex. RAPIDR has been extensively tested with a large sample set as part of the RAPID project in the UK. The package contains quality control steps to make it robust for use in the clinical setting.Availability and implementation: RAPIDR is implemented in R and can be freely downloaded via CRAN from here: information: Supplementary data are available at Bioinformatics online.
    Bioinformatics 07/2014; 30(20). DOI:10.1093/bioinformatics/btu419 · 4.98 Impact Factor
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