© 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?

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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

© Copyright 2009 by the American Association for Clinical Chemistry

DiscussionDiscussion

Application to Trisomy 18 and Trisomy 13Need to improve the precision for measuring GR

of chromosomes 18 and 13