Non-invasive prenatal diagnosis using cell-free fetal DNA in maternal plasma.

Currently, prenatal diagnosis of fetal genetic abnormalities, including
aneuploidies and recessive genetic disorders, requires an invasive diagnostic
procedure, specifically chorionic villus sampling or amniocentesis, to obtain
fetal material for genetic testing. Although the safety of these invasive
procedures has improved since their introduction, these tests carry a risk of
iatrogenic fetal loss of about 1%, and are therefore reserved for pregnancies
considered to be at high risk for a fetal genetic abnormality (Tabor 1986,
Odibo 2008a, Odibo 2008b, Tabor 2010).

The risk of aneuploidy is assessed by prenatal screening, nowadays offered to
all pregnant women. Risk calculation is based on maternal age, nuchal
translucency measurement by sonography and the serum markers
pregnancy-associated plasma protein-A (PAPP-A) and free beta hCG (free β-hCG).
This first trimester combination test can identify 85-90% of affected fetuses.
Of all women participating, 5-6% is screen-positive (also referred to as
false-positive, because the vast majority of fetuses in this group are normal)
and therefore offered an invasive diagnostic procedure. Since the screening
test only detects epiphenomena rather than the core pathology of chromosomal
abnormalities, it lacks diagnostic power (Snijders 1998, Wald 1999, Wald 2003,
Malone 2005, Kagan 2008).

The discovery of the presence of cell-free fetal DNA (cff DNA) in the plasma of
pregnant women has opened up possibilities for non-invasive prenatal diagnosis
(NIPD) (Lo 1997). DNA of fetal origin is detectable early in pregnancy in
amounts of approximately 10% of the total DNA circulating in maternal plasma
(Lo 1998, Lun 2008). The cff DNA is cleared from the maternal bloodstream
within hours after birth; thus, misdiagnosis resulting from remaining DNA from
a previous pregnancy is unlikely (Lo 1999). Detection of fetal-specific DNA
sequences is already used successfully for non-invasive determination of fetal
sex in women carrying a sex-linked recessive genetic disorder (Zhong 2000,
Rijnders 2001, Scheffer 2010) and of fetal Rhesus D genotype in Rhesus D
negative women (Faas 1998, Lo 1998, Bianchi 2005, Finning 2008, Scheffer 2011).

Unfortunately, detection of fetal genetic abnormalities, including aneuploidies
and recessive genetic disorders, from cff DNA is far more complicated. Fetal
DNA represents only a minor fraction of total DNA in maternal plasma, and is
therefore substantially diluted by the maternal DNA contribution. However,
using massively parallel shotgun sequencing (MPSS), it is possible to sequence
the first bases of millions of DNA fragments in maternal plasma to determine
their specific chromosomal origin (Chiu 2008, Fan 2008). Using this technique,
even relatively small changes in the representation of chromosomes, from a
fetus carrying three rather than two copies of a chromosome, can be detected
with statistically significant power. The results of recently published studies
using MPSS to detect fetal trisomy 21 in high-risk populations showed a
combined sensitivity and specificity of 99.1% (334/337) and 99.7% (2020/2027),
respectively (Chiu 2011, Ehrich 2011, Palomaki 2011).

Furthermore, research has shown the presence of cff RNA in maternal plasma as
well. In due time, it could be possible to monitor the unborn child and the
course of pregnancy through observation of fetal expression of certain genes
through the analysis of cff RNA in maternal plasma.

Previously, as described in phase 1 of the approved CMO protocol 2008/349 *Niet-
invasieve prenatale diagnostiek naar Down syndroom met gebruik van DNA en/of
RNA in het maternale plasma*, blood samples from pregnant women scheduled for
an invasive procedure have been collected, processed and stored in our center,
starting from March 2010. Whenever possible, a blood sample from their (male)
partners was collected, processed and stored as well. As described in phase 2
of the approved protocol, 52 of the stored maternal plasma samples have been
used for a pilot study of NIPD through MPSS of cff DNA in maternal plasma.
Results are very promising: all fetal aneuploidies for chromosomes 13, 18 and
21 and sex chromosomal aberrations could be reliably detected, with 100%
sensitivity and specificity (Faas 2012, in press).

The ultimate goal of this research is the clinical implementation of a
non-invasive test for prenatal fetal genotyping, including fetal sex
determination, using cff DNA in maternal plasma, to replace the current
standard practice of invasive tests, specifically chorionic villus sampling or
amniocentesis.

Available data from literature and in-house testing show that the application
of MPSS of cff DNA in maternal plasma for NIPD of fetal aneuploidies is robust
and sensitive, with diagnostic accuracy of this method even approaching that of
invasive tests. However, before clinical implementation of NIPD of fetal
aneuploidies and other fetal genetic abnormalities is feasible, extended
studies are needed to further improve and validate this new technique in our
own laboratory, and to study its performance in clinical practice.

This study aims to address the following primary objectives:
*- developing a standardized protocol, including quality control parameters,
for the use of NIPD by MPSS of cff DNA;
*- determine the sensitivity and specificity of NIPD by MPSS of cff DNA;
*- optimize throughput, turnaround time and costs;
*- determine the predictive values of NIPD by MPSS of cff DNA when used in a
clinical setting.

To be able to address the goal and objectives stated above, plasma and DNA
fractions of both pregnant women and their (male) partners are required. This
study therefore aims to collect, process and store maternal and paternal plasma
and DNA fractions, to be used for the further development and validation of a
non-invasive test for prenatal fetal genotyping, including fetal sex
determination.

The study will be a prospective, multicenter cohort study, consisting of
multiple phases.

Phase 1: technical and logistic optimization
The first phase of this study is aimed to develop a standardized protocol for
NIPD by MPSS of cff DNA in maternal plasma. Methods to demonstrate the presence
of cff DNA and measure the amount and fraction of cff DNA present will be
further developed and validated. Quality control parameters will be
established. Parts of laboratory and data analysis will be automated.
Furthermore, the logistics necessary to collect and analyze plasma samples, and
to document and distribute test results on a large scale, throughout the region
will be set up.

Phase 2: prospective validation
The second phase of this study is aimed to assess the performance of NIPD of
fetal aneuploidies using cff DNA in maternal plasma in a clinical setting,
applying the standardized protocol developed in phase 1 of this study.

In some female participants, and the vast majority of male participants, the
drawing of a study blood sample will not be able to be combined with an already
scheduled venipuncture as part of standard prenatal care. These participants
will have an additional venipuncture. The risks associated with this procedure
are negligible.

Participants will not benefit from their partaking in this study in any way.