Prenatal Screening Using Free DNA
in Maternal Blood
Jacob Canick, PhD
Alpert Medical School of Brown University
Women & Infants Hospital
Providence, RI, USA
Department of Pathology
Montefiore Medical Center
Bronx, NY
March 17, 2011
Declaration of Interests Pertinent
to this Discussion
Funding from SEQUENOM, Inc., San Diego, CA,
to conduct a clinical study on tests for trisomy 21
in pregnancy using fetal nucleic acids in maternal
plasma.
Current Screening Uses Prenatal Markers of the
Down Syndrome Phenotype
• The best test performance is currently:
95% DR @ 5% FPR
or
Full/Sequential Integrated Test
90% DR @ 2% FPR
85% DR @ 5% FPR
1st Trim. Combined or
Serum Integrated Test
80% DR @ 5% FPR
2nd Trim. Quad Test
• All screen positive women should be counseled on risks and
benefits of invasive procedures for karyotype analysis.
Current Screening for fetal Down Syndrome
using Phenotypic or “Surrogate” Markers
Fetal ultrasound:
Maternal Serum:
PAPP-A
low
Nuchal Translucency
increased
AFP
low
Nasal Bone
absent/small
uE3
low
Nuchal fold thickness
larger
Femur/Humerus
shorter
b-hCG
elevated
inhibin A
elevated
Echogenic cardiac focus present
Ductus venosus doppler reversed a-wave
Nuchal Translucency
Nasal Bone
Ductus venosus doppler
www.fetalmedicine.com/fmf
Future Prenatal Testing:
Prenatal Screening and Diagnosis of Fetal Trisomies
New direction: direct identification of the disorder (markers of
genotype rather than markers of phenotype).
Targeted to the specific numerical chromosomal disorder:
Trisomy 21 rather than Down syndrome phenotype
Trisomy 18 rather than Edwards syndrome phenotype
Trisomy 13 rather than Patau syndrome phenotype
Measure specific free fetal nucleic acids (DNA or RNA) in
the maternal circulation.
Background:
Fetal Nucleic Acids in Maternal Plasma
• First report of free fetal DNA in maternal
circulation. (Lo YMD et al. Lancet
1997;350:485-7)
• Fetal DNA clears rapidly from maternal
circulation after the baby is delivered. (Lo
YMD et al. Am J Hum Genet 1999;64:218-24)
• First report of free fetal RNA in maternal
circulation. (Poon LLM et al. Clin Chem
2000;46:1832-4)
• Prenatal diagnosis of fetal RHD status by
molecular analysis of maternal plasma. (Lo
YMD et al. N Engl J Med 1998;339:1734-8)
Cell-free DNA in the Maternal Circulation
Placenta
Maternal plasma
Maternal blood cells
• Both cell-free fetal and cell-free maternal DNA circulate in
maternal plasma.
• Cell-free fetal and maternal DNA circulate in maternal plasma
as relatively short fragments (150-200 base pairs) and
represent the entire genome.
• Fetal DNA comes primarily from the placenta.
• Maternal DNA comes primarily from maternal blood cells.
• Fetal DNA is 5-25% of the total cell-free DNA (~10% on
average).
Potential clinical applications of analysing fetal nucleic acids
in maternal plasma. Lo and Chiu, Nature Reviews Genetics 2007
Massively Parallel Sequencing
(MPS):
Identifying Down syndrome using
circulating cell free DNA in maternal
plasma
First publications on MPS for trisomy 21 detection
PNAS 2008;105:15255
PNAS 2008;105:20458
The Concept
10% of free DNA in maternal plasma is fetal
Relative amount of chromosome 21
Normal Mother Normal Fetus
18 copies
+
2 copies
20 copies
Relative amount of chromosome 21
Normal Mother Down syndrome Fetus
18 copies
+
3 copies
21 copies
Need to distinguish 21 copies from 20 copies, a 5% difference.
(assumes 10% of ccfDNA is fetal)
But, fetal and maternal DNA
are not distinguishable by MPS
Relative amount of chromosome 21
18 copies
+
2 copies
20 copies
Relative amount of chromosome 21
18 copies
+
3 copies
21 copies
Need to distinguish 21 copies from 20 copies, a 5% difference.
(assumes 10% of ccfDNA is fetal)
Schematic illustration of the procedural framework for using
massively parallel genomic sequencing for the noninvasive
prenatal detection of fetal chromosomal aneuploidy.
Chiu R W K et al. PNAS 2008;105:20458
Schematic illustration (con’t)
%chr21 = 1 / 51 = 2%
Chiu R W K et al. PNAS 2008;105:20458
Schematic illustration (con’t)
• For each chromosome, determine its average % of unique
sequences, compared to the total number of sequences in the
normal human genome.
• Do this by getting data from many ‘normal’ samples.
• This will produce a normal distribution (mean ± SD) for each
chromosome.
• For example:
% unique sequences in chromosome 21 in six different euploid genomes
2.01
2.00
1.98
2.02
2.03
1.99
2.01 ± 0.02 (mean ± standard deviation)
Schematic illustration (con’t)
mean
.… -6
-5
-4
4
5
6….
Z score (± SD)
schematic from www.sci.sdsu.edu
Schematic illustration (con’t)
To test an individual:
Determine the % of chromosome 21 unique sequences for that
person and compare that % to the mean, in terms of ± SD (Z Score).
Z score
calculation
% unique sequences in chromosome 21
in euploid
2.01
2.00
1.98
2.02
2.03
1.99
-----2.01 ± 0.02
in test sample
Z = (2.11 – 2.01)
0.02
2.11
Z=
0.10
0.02
Z=
5
Chiu R W K et al. PNAS 2008;105:20458
Schematic illustration (con’t)
euploids
trisomy 21 cases
Chiu R W K et al. PNAS 2008;105:20458
How is this implemented?
Four steps in the MPS process
1. Library Preparation
• Purify free DNA from maternal plasma (already
fragmented)
• Add special adapters to both ends
• Dilute to get proper concentration range
2. Cluster Generation
• Run samples through Illumina flow cell (8 lanes per cell)
to capture fragments
• Solid-phase amplification of fragments to generate
clusters
1
2
3
4
Four steps in the MPS process
3. Sequencing by Synthesis
•
•
•
•
Illumina High Seq 200, a pumping and imaging system
Sequence the first 36 bases
>10 million clusters sequenced per flow cell lane
>1 terabyte of data per flow cell
4. Data Analysis
• Alignment (chromosome matching) using human genome
database
• One matching error per 36 bases allowed
• Interpretation of results:
% of matches on chromosome 21
Z score for each sample
Published results so far…
Proportion of unique sequences per chromosome,
from three plasma samples and genome database
Unique matches (%)
Bars (Left to Right)
Expected genomic %
Normal female fetus
Dup NFF, protocol 2
Normal male fetus
Dup NMF, protocol 2
Mix of 2 norm males
Dup Mix, protocol 2
Chromosome Number
Chiu R W K et al. PNAS 2008;105:20458
Proportion of unique sequences per chromosome,
from three plasma samples and genome database
Bars (Left to Right)
Expected genomic %
Normal female fetus
Dup NFF, protocol 2
Normal male fetus
Dup NMF, protocol 2
Mix of 2 norm males
Dup Mix, protocol 2
Chiu R W K et al. PNAS 2008;105:20458
Black
Blue
Orange
Green
Red
genomic representation
normal male
normal female
T21 male
T21 female
Z-score
% of all unique reads
Percent unique reads and corresponding z-score for
chromosome 21, on 28 maternal plasma samples
Normal range
Chiu R W K et al. PNAS 2008;105:20458
Z scores for each chromosome
New publications on MPS for trisomy 21 detection
Chiu et al.
8-plex
86 cases
571 controls
DR: 79%
FPR: 1%
2-plex
86 cases
146 controls
DR: 100%
FPR: 2%
Ehrich et al.
monoplex
39 cases
410 controls
DR: 100%
FPR: 0.3%
Independent Clinical Trial
Nearing Completion
BROWN
Women & Infants’
• Enrolled pregnant women, from 27 Recruitment Sites
worldwide, were at high risk based on prenatal screening,
abnormal fetal ultrasound, age >38 years.
• All enrollees had maternal plasma samples taken prior to
CVS or amniocentesis; sample processing within 6 hours.
• More than 4500 women enrolled, with more than 200
cases of fetal trisomy 21 (half 1st trim, half 2nd trim.)
• Other aneuploidies are also studied.
• Testing of coded samples by Massively Parallel
Sequencing of free DNA in the maternal plasma at SCMM.
• Funded by Sequenom Inc.
Free DNA-based Testing for Trisomy 21:
Further Issues
• Cost
 hundreds, thousands of $$$$?
 getting less expensive very quickly
• Turnaround time
 3 days, 7 days, longer?
• Availability
 limited lab sites
 intellectual property issues
• Amnio/CVS still necessary?
 Is it diagnostic, or just a very good screening test?
Conclusions
• Current methods of prenatal screening reach a
performance of 90% DR at a 5% FPR.
• Measurement of free DNA in the maternal circulation
holds the possibility for considerably better screening
performance, perhaps even non-invasive diagnosis.
• Currently, massive genomic sequencing appears to hold
the most promise.
• Other chromosomal aneuploidies should be able to be
identified by this approach.
• Other genetic defects, including single gene disorders,
may also be identified by this approach.
Fetal DNA Study Collaborators
Women & Infants Hospital/Brown University:
Glenn Palomaki, PhD
Ed Kloza, MS
Geralyn Lambert-Messerlian, PhD
Regina Traficante, PhD
UCLA School of Medicine:
Stan Nelson, MD
Wayne Grody, MD, PhD
Sequenom Center for Molecular Medicine:
Mathias Ehrich, MD
Dirk van den Boom, PhD
Allan Bombard, MD
and investigators at 27 sites in NA, SA, Europe, Australia