© Copyright 2009 by the American Association for Clinical Chemistry
Maternal Plasma DNA Analysis Using Massively Parallel Sequencing-By-Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21
Rossa W.K. Chiu, Hao Sun, Ranjit Akolekar, Christopher Clouser, Clarence Lee, Kevin Mckernan, Daixing Zhou, Kypros H. Nicolaides, and Y. M. Dennis Lo
March 2010
http://www.clinchem.org/cgi/content/full/56/3/460
© Copyright 2010 by the American Association for Clinical Chemistry
Journal ClubJournal Club
© Copyright 2009 by the American Association for Clinical Chemistry
IntroductionIntroduction
Prenatal DiagnosisA part of obstetrics care
Fetal tissue for genetic analysis is obtained conventionally by amniocentesis or chorionic villus sampling
The procedures are invasive with risk of fetal miscarriage
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction (cont)Introduction (cont)Fetal DNA in Maternal Plasma
First reported in 1997
Exists as short fragmented molecules in cell-free form
Only amounts to ≈ 10% of the total DNA among a background of maternal DNA
Successfully used for noninvasive prenatal assessment of fetal sex and fetal rhesus D status
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction (cont)Introduction (cont)
Trisomy 21Typically due to presence of a third copy of chromosome (chr) 21 in fetal genome
Incidence ≈ 1 out of 800 pregnancies
Other aneuploidies: trisomy 18, trisomy 13, Turner syndrome
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion
Why is it more challenging to achieve noninvasive prenatal diagnosis of fetal trisomy 21 by fetal DNA analysis in maternal plasma compared with applications such as fetal sex determination?
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and MethodsMaterials and Methods
Massively Parallel Genomic SequencingPlasma was obtained from pregnant women
DNA was extracted
Universal adaptors were added to the ends of the plasma DNA molecules
Clonal amplification by emulsion PCR
Random sequencing of DNA using sequencing-by-ligation
50 bp from one end of each plasma DNA molecule sequenced
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and Methods (cont)Materials and Methods (cont)
Bioinformatics Analysis35 bp of each sequenced read was mapped to the repeat-masked reference human genome
Only reads that perfectly mapped to one location of the reference genome were included for further analysis
Chromosomal origin of each included read was recorded
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and Methods (cont)Materials and Methods (cont)
Chromosomal Genomic Representation (GR)GR of chrN = %chrN = # reads from chrN / # total reads
Expected GR of chrN = nucleotide content of chrN as a proportion of the genomic content of a normal human genome
Deviation from Expected GR (experimentally derived GR for chrN) – (expected GR for chrN) expected GR for chrN
CV of GRSD of GR for chrN / mean GR for chrN
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and Methods (cont)Materials and Methods (cont)
Diagnosis of Trisomy 21
>chr21 z scores%chr21test case – %chr21reference controls
SDreference controls
z score > 3 used as diagnostic cutoff to identify trisomy 21
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion
What factors may cause a deviation in the measured GR from the expected GR for any particular chromosome?
© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry
Figure 1. z scores for chromosome 21 in the control (cases 1–4, in black), euploid (cases 5–10, in blue), and trisomy 21 (cases 11–15, in red) pregnancies. A z score of 3 (dashed line) was used as a cutoff to determine the presence of overrepresentation of sequences from chromosome 21.
ResultsResults
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Figure 2. Bar chart showing the median degree of deviation in genomic representation for the human chromosomes ordered from left to right in increasing GC contents.
ResultsResults
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Figure 3. CV for measuring the %GR of each chromosome ordered from left to right in increasing GC
contents.
ResultsResults
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion
What factors may affect the z score values and its ability in discriminating trisomy 21 from euploid cases?
© Copyright 2009 by the American Association for Clinical Chemistry
DiscussionDiscussion
Noninvasive Prenatal Diagnosis of Trisomy 21Accurate diagnosis can be achieved by massively parallel sequencing of maternal plasma DNA
Pending validation by large-scale studies
Pending reduction in sequencing costs and improvements in throughput