Differential DNA Methylation as a Tool for Noninvasive Prenatal Diagnosis (NIPD) of X Chromosome Aneuploidies

Institute of Genetics and Biophysics A. Buzzati Traverso, Naples, Italy.
The Journal of molecular diagnostics: JMD (Impact Factor: 4.85). 11/2010; 12(6):797-807. DOI: 10.2353/jmoldx.2010.090199
Source: PubMed


The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples.

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Available from: Maurizio D'Esposito, Oct 13, 2015
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    • "The greatest advantage of a whole genome approach is that it enables the impartial analysis of the entire genome, enabling detection of genomic aberrations without a priori region selection. Region-specific methods theoretically support higher throughput while still achieving acceptable performance for the two most common trisomies (trisomy 21 and trisomy 18); however, these targeted assays, including those not based on Massively Parallel Sequencing (MPS) [10]–[12], are restricted by the significant amount of re-development required when additional content, for example less frequent trisomies or sex chromosome aneuploidies, is introduced. Additionally, it remains to be seen how well these targeted methods can identify events such as partial trisomies or other large copy number variations. "
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    ABSTRACT: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.
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    ABSTRACT: The conventional methods of prenatal diagnosis are being challenged by recent technologies. Chromosomal microarrays already in mainstream use for postnatal genetic diagnosis are increasingly used for prenatal diagnosis, mainly in pregnancies with sonographic anomalies but also for routine screening after any invasive procedure. Arrays have demonstrated the ability to detect submicroscopic copy number variations, providing an approximately 2.1 % increase in the detection rate of pathogenic copy number variations regardless of the referral indication, and rising to an approximately 5.3 % increase above conventional karyotyping in the presence of sonographic anomalies. More recently, novel technologies and methods of non-invasive prenatal testing are reaching clinical applications beyond fetal sex determination and rhesus blood group genotyping. Massively parallel sequencing methods have been shown to confidently detect trisomy 21 from cell-free DNA isolated from a maternal plasma sample and are rapidly entering clinical use. Targeted methods including epigenetic differences between the fetal and maternal genomes such as differential methylation are also being applied for non-invasive aneuploidy detection. It can be anticipated that very soon chromosomal microarrays will become the first-tier test for invasive prenatal diagnosis. In addition, we believe that non-invasive prenatal testing will gradually replace the need for invasive prenatal diagnosis with the associated risk of pregnancy loss.
    06/2013; 1(2). DOI:10.1007/s40142-013-0016-4
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