Transcript Journal Club - Clinical Chemistry

1
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 2013
www.clinchem.org/content/59/11/1583.full
© Copyright 2013 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
Genomewide
methylation
profile of the
first trimester
samples
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 1Mb 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.
© 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;59:1547-9.
© Copyright 2009 by the American Association for Clinical Chemistry
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