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This article discusses the challenges and potential of noninvasive prenatal diagnosis of fetal trisomy 21 using fetal DNA analysis in maternal plasma. It explores the use of massively parallel genomic sequencing and bioinformatics analysis for accurate diagnosis. The factors affecting genomic representation and z-score values are also examined. Further applications to trisomy 18 and trisomy 13 are discussed.
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Introduction • 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
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
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
Question • 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?
Materials 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
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
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
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
Question • What factors may cause a deviation in the measured GR from the expected GR for any particular chromosome?
Results Figure 1. zscores 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.
Results 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.
Results Figure 3. CV for measuring the %GR of each chromosome ordered from left to right in increasing GC contents.
Question • What factors may affect the z score values and its ability in discriminating trisomy 21 from euploid cases?
Discussion • 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
Discussion • Application to Trisomy 18 and Trisomy 13Need to improve the precision for measuring GR of chromosomes 18 and 13
