Noninvasive Prenatal Methylomic Analysis by Genomewide Bisulfite Sequencing of Maternal Plasma DNA F.M.F. Lun, R.W.K. Chiu, K. Sun, T.Y. Leung, P. Jiang, K.C.A. Chan, H. Sun, and Y.M.D. Lo November © Copyright 2013 by the American Association for Clinical Chemistry 1

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  Epigenetics  Refers to cellular and biological mechanisms that alter gene expression or the expressed function of DNA without changing the DNA sequence.  Examples of epigenetic mechanisms include DNA methylation, histone modifications, chromatin remodeling and noncoding RNAs.  DNA methylation  Is one of the most commonly studied epigenetic phenomenona and is mostly in the form of cytosine methylation. This refers to the addition of a methyl group to the 5' carbon of cytosine residues in CpG dinucleotides.

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  DNA methylation and development  DNA methylation plays a role in the control of gene expression, gene promoter function, X chromosome inactivation and genomic imprinting.  Researchers have much interest in studying the role of DNA methylation in fetal growth and development as well as patterns of aberrant methylation related to fetal or pregnancy-associated pathologies.  However, fetal or placental tissues are not readily available from viable pregnancies before term.

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  Circulating cell-free fetal DNA  During pregnancy, DNA mostly from dying placental cells are released into the mother’s blood. Such fetal DNA molecules are found among a major background of the mother’s DNA in maternal plasma.  The circulating fetal DNA molecules are cell-free in nature and exist as short fragments.  Cell-free fetal DNA therefore is a source of fetal genetic material that could be sampled noninvasively through the collection of a mother’s blood sample.

© Copyright 2009 by the American Association for Clinical Chemistry Question  There has been a large body of work on studying the DNA methylation profile of placental tissues  What have researchers found to date?  What impact do abnormalities in DNA methylation have on the development of the pregnancy, the fetus, or the offspring?

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Noninvasive fetal and placental methylomic analysis  Peripheral blood samples were collected from a pregnant woman in the first trimester, third trimester and after delivery.  Maternal plasma DNA was subjected to massively parallel bisulfite sequencing.  Bioinformatics procedures were applied to distinguish the fetal and placental methylation profiles from that of the background maternal DNA.

© Copyright 2009 by the American Association for Clinical Chemistry Question  Describe the two different approaches used in this study to determine the fetal / placental methylation profile from maternal plasma despite the presence of the large background of maternal DNA

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Trisomy 21 detection by plasma DNA bisulfite sequencing  Peripheral blood samples were collected from pregnant women who presented for antenatal Down syndrome screening.  Maternal plasma DNA was subjected to massively parallel bisulfite sequencing.  The methylation density of plasma DNA molecules derived from chromosome 21 was normalized by the methylation densities of other chromosomes.

© Copyright 2009 by the American Association for Clinical Chemistry Results  Noninvasive genomewide profiling of the placental methylome  Through the analysis of maternal plasma, the placental tissue methylation profile was successfully determined in a genomewide manner.  The noninvasive nature of this approach enabled one to serially monitor the changes of the placental tissue methylation profile throughout pregnancy as shown for the studied pregnancy.  Placental DNA was shown to be hypomethylated.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Circos plots of methylation density per 1-Mb bin. Chromosome ideograms (outermost ring) are oriented pter-qter in a clockwise direction (centromeres are shown in red). The second outermost track shows the number of CpG sites in the corresponding 1-Mb regions up to 20,000 sites. The methylation densities of the corresponding 1- Mb regions are shown in the other tracks based on the color scheme shown in the center. From inside to outside: chorionic villus sample, fetal-specific reads in maternal plasma, shared reads in maternal plasma, combined fetal and non-fetal reads in maternal plasma, and maternal blood cells. Genomewidemethylation profile of the first trimester samples

© Copyright 2009 by the American Association for Clinical Chemistry Results  Applications  The study further shows that the data enabled the noninvasive assessment of trisomy 21, the imprinting status of genes, gestational-age dependent changes in DNA methylation of gene loci, relationship between DNA methylation and the size of plasma DNA fragments, and the noninvasive search for placental tissue specific methylation markers.  Other potential applications are also described in the Editorial commentary that accompanies the article.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Trisomy 21 detection. Plot of normalized chromosome 21 methylation densities of maternal plasma samples collected from euploid and trisomy 21 pregnancies.

© Copyright 2009 by the American Association for Clinical Chemistry Table 2. Numbers of placental tissue differentially methylated loci predicted from the maternal plasma DNA sequencing data.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Methylation densities and sizes of plasma DNA molecules. First trimester maternal plasma. Data for all the sequenced reads that covered at least one CpG site are represented by the blue curve. Data for reads that also contained a fetal-specific SNP allele are represented by the red curve. Data for reads that also contained a maternal-specific SNP allele are represented by the green curve.

© Copyright 2009 by the American Association for Clinical Chemistry Question  What other applications could one explore with the methodologies developed in this study?

© Copyright 2009 by the American Association for Clinical Chemistry Accompanying Editorial: Oudejans C.B.M. Maternal Plasma Bisulfite DNA Sequencing: Tomorrow Starts Today. Clinical Chemistry 2013;59:

© Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at Download the free Clinical Chemistry app on iTunes for additional content! Follow us