Prenatal Screening Using Free DNA

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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
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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.
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Current Screening Uses Prenatal Markers of the
Down Syndrome Phenotype

• The best test performance is currently
• 95 DR _at_ 5 FPR
• or Full/Sequential
  Integrated Test
• 90 DR _at_ 2 FPR
• 85 DR _at_ 5 FPR 1st Trim. Combined or
  Serum Integrated Test
• 80 DR _at_ 5 FPR 2nd Trim. Quad Test
• All screen positive women should be counseled on
  risks and benefits of invasive procedures for
  karyotype analysis.

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Current Screening for fetal Down Syndrome using
Phenotypic or Surrogate Markers
Fetal ultrasound Nuchal Translucency
increased Nasal Bone absent/small
Nuchal fold thickness larger
Femur/Humerus shorter Echogenic cardiac
focus present Ductus venosus doppler
reversed a-wave
Maternal Serum PAPP-A low
AFP low uE3 low
b-hCG elevated inhibin A elevated
Nasal Bone
Nuchal Translucency
Ductus venosus doppler
www.fetalmedicine.com/fmf
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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.

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Background Fetal Nucleic Acids in Maternal Plasma

• First report of free fetal DNA in maternal
  circulation. (Lo YMD et al. Lancet
  1997350485-7)
• Fetal DNA clears rapidly from maternal
  circulation after the baby is delivered. (Lo YMD
  et al. Am J Hum Genet 199964218-24)
• First report of free fetal RNA in maternal
  circulation. (Poon LLM et al. Clin Chem
  2000461832-4)
• Prenatal diagnosis of fetal RHD status by
  molecular analysis of maternal plasma. (Lo YMD et
  al. N Engl J Med 19983391734-8)

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Cell-free DNA in the Maternal Circulation

• 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).

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Potential clinical applications of analysing
fetal nucleic acids in maternal plasma. Lo and
Chiu, Nature Reviews Genetics 2007
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Massively Parallel Sequencing (MPS)
Identifying Down syndrome using circulating cell
free DNA in maternal plasma
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First publications on MPS for trisomy 21 detection
PNAS 200810520458
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The Concept 10 of free DNA in maternal plasma
is fetal
Relative amount of chromosome 21 Normal Mother
Normal Fetus
Relative amount of chromosome 21
Normal Mother Down syndrome Fetus
18 copies 2 copies 20 copies
18 copies 3 copies 21 copies
Need to distinguish 21 copies from 20 copies, a
5 difference. (assumes 10 of ccfDNA is fetal)
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But, fetal and maternal DNA are not
distinguishable by MPS
Relative amount of chromosome 21
Relative amount of chromosome 21
18 copies 2 copies 20 copies
18 copies 3 copies 21 copies
Need to distinguish 21 copies from 20 copies, a
5 difference. (assumes 10 of ccfDNA is fetal)
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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 200810520458
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Schematic illustration (cont)
chr21 1 / 51 2
Chiu R W K et al. PNAS 200810520458
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Schematic illustration (cont)

• 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)
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Schematic illustration (cont)
mean
4 5 6.
. -6 -5 -4
Z score ( SD)
schematic from www.sci.sdsu.edu
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Schematic illustration (cont)
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).
unique sequences in chromosome 21 in euploid
in test sample
2.01 2.00 1.98 2.02 2.03 1.99 ------ 2.01 0.02
2.11
Chiu R W K et al. PNAS 200810520458
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Schematic illustration (cont)
Chiu R W K et al. PNAS 200810520458
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How is this implemented?
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Four steps in the MPS process

• Library Preparation
• Purify free DNA from maternal plasma (already
  fragmented)
• Add special adapters to both ends
• Dilute to get proper concentration range
• 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
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Four steps in the MPS process

• Sequencing by Synthesis
• Illumina High Seq 200, a pumping and imaging
  system
• Sequence the first 36 bases
• gt10 million clusters sequenced per flow cell lane
• gt1 terabyte of data per flow cell
• 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

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Published results so far
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Proportion of unique sequences per chromosome,
from three plasma samples and genome database
Unique matches ()
Chromosome Number
Chiu R W K et al. PNAS 200810520458
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Proportion of unique sequences per chromosome,
from three plasma samples and genome database
Chiu R W K et al. PNAS 200810520458
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Percent unique reads and corresponding z-score
for chromosome 21, on 28 maternal plasma samples
of all unique reads
Black genomic representation Blue normal
male Orange normal female Green T21
male Red T21 female
Z-score
Normal range
Chiu R W K et al. PNAS 200810520458
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Z scores for each chromosome
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New publications on MPS for trisomy 21 detection
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8-plex 86 cases 571 controls
DR 79 FPR 1
2-plex 86 cases 146 controls
DR 100 FPR 2
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monoplex 39 cases 410 controls
DR 100 FPR 0.3
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• Enrolled pregnant women, from 27 Recruitment
  Sites worldwide, were at high risk based on
  prenatal screening, abnormal fetal ultrasound,
  age gt38 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.

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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?

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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.

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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