Next-Generation–Sequencing-Based Noninvasive Prenatal Diagnosis - Next Generation Sequencing Technologies in Medical Genetics - page 42

2009 ; Avent and Chitty 2006 ; Bianchi et al. 2005 ; Hahn et al. 2011 ; van der Schoot
et al. 2003 ). NIPD has been more challenging for conditions in which analysis of
maternal alleles is required owing to the ubiquitous presence of free maternal DNA
in plasma. This situation still represents the most important technical obstacle to
achieving routine NIPD for the common chromosomal abnormalities, such as
aneuploidy.
In 2008, however, there appeared a brace of reports that applied the emerging
next-generation-sequencing (NGS) technologies to the analysis of trisomies with
DNA extracted from maternal plasma (Chiu et al. 2008 ; Fan et al. 2008 ). Several
groups confi rmed this breakthrough, and last years investigation of a very large
cohort proved the effi cacy of NGS as applied to chromosome counting in maternal
plasma samples (Chiu et al. 2011 ). Several consortia [e.g., the European Commission
consortium EuroGentest ( http://www.eurogentest.org ) and the UK RAPID consor-
tium ( http://www.rapid.nhs.uk )] are driving the implementation of this technology
into clinical practice. Thus, in this chapter we explore the exquisite resolution of
NGS and its ability to sequence a population of DNA molecules to defi ne chromo-
some copy number following the introduction of the common aneuploidy T21.

5.2 Trisomy 21, a Common Aneuploidy

Trisomy 21 (T21) is the most common chromosomal abnormality in live-born chil-
dren. By employing invasive testing, the diagnosis can be determined early in preg-
nancy with a high risk of miscarriage (Lo et al. 2007 ). These tests are routinely for
women at increased risk for fetal trisomy based on maternal age, serum markers for
trisomy, and ultrasound measurement of the fetal nuchal translucency (Driscoll
et al. 2009 ). From screening program data, one in every 20 women is offered inva-
sive testing (Wapner et al. 2003 ). Recently, NIPD, through the use of cffDNA, has
been employed to the diagnosis of T21 (Lo et al. 2007 ). However, due to the vari-
ability (1-10 %) of percentage of cffDNA in maternal plasma, it remains challeng-
ing to detect fetal sequences in a large pool of maternal DNA (Hahn et al. 2011 ).
Therefore, a number of methods have been developed and used in the fi eld of non-
invasive T21, including the NGS, and are described in the following section.

5.3 Methods for the Detection of cffDNA

The scarcity of cffDNA in maternal blood and its coexistence with maternal DNA
represent the two major limitations for the use of cffDNA for diagnosis. Both mater-
nal plasma and serum contain cffDNA; however, plasma is the material of choice
for prenatal diagnosis since it contains less maternal background DNA. Various
methods have been used to overcome the presence of maternal background cell-free
DNA (cfDNA), including methods based on the size difference of maternal