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OBSTETRICS AND GYNECOLOGY ADVANCES

PRENATAL AND MATERNAL


DIAGNOSIS, SCREENING AND
INFANT DEVELOPMENT
IMPLICATIONS

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OBSTETRICS AND
GYNECOLOGY ADVANCES
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OBSTETRICS AND GYNECOLOGY ADVANCES

PRENATAL AND MATERNAL


DIAGNOSIS, SCREENING AND
INFANT DEVELOPMENT
IMPLICATIONS

AARON HENDERSON
EDITOR

New York

Copyright 2015 by Nova Science Publishers, Inc.


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CONTENTS
Preface
Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Index

vii
Maternal Smoking in Pregnancy and Its Impact
on Placental and Fetal Development
Emilio Gonzlez-Jimnez

Maternal Smoking during Pregnancy and the Risk


of Sleep Disturbances in Infants
Igor A. Kelmanson

Prenatal Screening and Diagnosis of Facial Clefts:


Where Are We Now?
William W. K. To and Meliza C. W. Kong

17

Noninvasive Prenatal Diagnosis by Sequencing


Circulating Fetal DNA
Liang Xu and Rui Shi

41

Array CGH in Prenatal Diagnosis


Paola Evangelidou, Philippos C. Patsalis
and Carolina Sisman

53

107

PREFACE
One of the challenging issues of contemporary pediatrics is potential longterm effects of maternal cigarette smoking during pregnancy on the subsequent
infant behavioral and cognitive development. The chapters in this book
address to what extent and in what direction maternal smoking during
pregnancy may influence infant's behavior during sleep. Recent reports were
highly indicative that sleep disturbances were common in the newborns of the
mothers who have been heavy smokers during pregnancy. Snuff consumption
during pregnancy is discussed, which has many adverse effects on the fetus,
i.e., spontaneous abortion, premature delivery premature sagging of the
placenta, vaginal bleeding, high blood pressure and heart rate, premature
rupture of membranes, among others.
The authors of the third chapter discuss orofacial clefts, and with the
development of ultrasound technology, how they can be detected antenatally
which can ultimately help with the planning postnatal management of babies.
Nowadays, prenatal diagnosis is necessary for pregnant women. In the
fourth chapter, technical advancements of using maternal circulating nucleic
acids as the sample in noninvasive studies is introduced, highlighting the
utilization of next-generation sequencing in the screening of genetic diseases.
The fifth and final chapter introduces Comparative Genomic Hybridization
(CGH) analysis in postnatal analysis and its use as a first-tier test in cases of
Intellectual Disabilities.
Chapter 1 The consumption of snuff is one of the major public health
problems in the world, and in economically developed countries. Today, Spain
is in phase III of the theoretical model of the tobacco epidemic, characterized
by increased consumption in women, a significant decrease in males and
increased attributable mortality in both sexes even higher among men.

viii

Aaron Henderson

Snuff smoke contains thousands of chemicals, specifically over 4,000


different components most harmful to health. The most studied components
are nicotine, tar and carbon monoxide resulting combustion carbon cigarette.
Nicotine is an alkaloid present in the leaf snuff with great addictive power and
speed given its ability to cross the blood brain barrier.
In recent years, there has been a reduction in the overall prevalence of
smoking; however, an increased consumption of snuff among younger women,
ie in the reproductive age groups is observed. In connection with the
consumption of snuff during pregnancy, in Spain, there is a high consumption
of snuff during pregnancy. Spanish population studies show a prevalence of
smoking among pregnant women ranges from 22% to 32%. However, despite
the consumption of snuff is a risk to the health of the fetus, not all pregnant
women manage to leave the snuff during pregnancy.
Snuff consumption during pregnancy has many adverse effects on the
fetus, ie, spontaneous abortion, premature delivery, placenta previa, premature
sagging of the placenta, vaginal bleeding, high blood pressure and heart rate,
premature rupture of membranes, among others.
Among the causes for the increased number of spontaneous abortions in
women smokers include placental malformations, mainly villous infarcts
associated with a deficit of oxygenation and placental blood flow. In addition,
smoking can cause a deficiency in vitamin absorption B once hydrocyanic
acid in cigarettes reduces the levels of this vitamin increases the risk of
preterm birth by 10-20%. Relative to premature rupture of membranes, the
mean relative risk of preterm birth for this cause doubles among women
smokers versus nonsmokers, being directly proportional to the number of
cigarettes smoked risk. In addition, these pregnant women who smoke often
have a greater number of infections in the amniotic fluid situation justifies
further cases of polyhydramnios in these pregnant women. For elevations of
blood pressure and heart rate, nicotine is its charge, stimulating the central
nervous system and releasing catecholamines from the adrenal medulla.
Chapter 2 One of the challenging issues of contemporary pediatrics is
potential long-term effects of maternal cigarette smoking during pregnancy on
the subsequent infant behavioural and cognitive development. Of particular
interest is the question whether, to what extent and in what direction maternal
smoking during pregnancy may influence infant's behaviour during sleep. This
chapter aimed to address the issue. Cigarette smoking is known to be
associated with sleep difficulties in the adults, and several animal experimental
studies suggest that the nicotine contained in cigarette smoke may interfere
with the normal maturation of sleep and wake. Recent reports were highly

Preface

ix

indicative that sleep disturbances were common in the newborns of the


mothers who have been heavy smokers during pregnancy. Changed sleep
integrity, sleep disordered breathing and altered arousal mechanisms observed
after exposure to tobacco smoke have been repeatedly implicated to the
mechanisms of sudden infant death syndrome. According to own findings, the
babies born to smoking mothers were more commonly reported as having
bedtime problems and irregular sleep schedule. The mechanisms underlying
changes in sleep due to prenatal tobacco exposure remain to be elucidated,
although it is known that they may be associated with profound alterations in
neurotransmitter disposition, evident in specific neuronal pathways and
persisting after birth.
Conclusion: Maternal smoking during pregnancy is one of the major
predictors of sleep disturbances in infants.
Chapter 3 Orofacial clefts are one of the most common non-syndromic
congenital structural abnormalities. In the past, congenital cleft lip and palate
are often only diagnosed after the baby was born. With developments in
ultrasound technology in recent years, the proportion of cleft lip/ palate that
can be detected antenatally has progressively increased. The wide-spread use
of second-trimester ultrasound screening for morphological abnormalities in
the recent two decades have allowed a very high proportion of significant cleft
lip as well as associated cleft palate defects to be diagnosed antenatally,
though the detection rate for isolated cleft palate (with intact lips) still remains
very low. When cleft lip is first detected on ultrasound examination, a
maternal-fetal medicine specialist will be responsible for performing further
detailed scanning to evaluate whether the facial cleft is an isolated defect, and
whether other genetic syndromes are likely to be associated with the condition.
While conventionally, two-dimensional scanning has been used for screening
of lip clefts, the development of three/ four-dimensional ultrasound scanning
technology has allowed more precise evaluation of the severity of the cleft lip
and the presence and absence of associated alveolar and palatal clefts. Various
three-dimensional scanning techniques to assess such defects have been
described in the literature, including the reverse face view, flipped face view,
oblique face view and so on, but there is still no consensus as to the most
effective and practical methods. In addition, there is still controversy as to
whether any of these techniques would have better performance than others.
As fetal magnetic resonance imaging gradually becomes an accessible
modality of imaging in modern obstetrics, it is likely to become an additional
tool to assess these defects. All these imaging techniques should yield useful

Aaron Henderson

information for antenatal counselling of the parents, and for planning postnatal
management of the babies.
Chapter 4 Nowadays, prenatal diagnosis is necessary for pregnant
women. For the parents who are expecting a child, the genetic test may
provide the information whether they are carrying rare gene mutations and
whether they are at risk of passing them onto their offspring. However, the
ultimate determination of genetic diseases often requires invasive procedures
such as amniocentesis and chorionic villus sampling, which may cause fetal
miscarriage. A noninvasive type of prenatal diagnosis needs to be developed in
clinical practice to dispel safety concerns. In this paper the authors will
introduce the technical advancement of using maternal circulating nucleic
acids as the sample in noninvasive studies, and highlight the utilization of
next-generation sequencing in the screening of genetic diseases.
Chapter 5 Chromosomal abnormalities are the cause of many human
genetic disorders. Karyotyping has been for decades the golden standard
method for prenatal diagnosis and for the diagnosis of numerical and large
structural abnormalities (<3-10Mb). These, if present may result in congenital
anomalies, dysmorphism, intellectual disability, autism or other neurological
abnormalities. With the introduction of array Comparative Genomic
Hybridization (CGH) analysis in postnatal analysis and its use as a first-tier
test in cases of Intellectual disabilities, it has been postulated that it might also
become the first-tier test in prenatal diagnosis as well.
rray CGH, a high throughput, comprehensive and fast technology, has
proven its ability to detect subtle copy number changes that can go undetected
by light microscopy. Array CGH is a valuable tool for the determination of
copy number changes in children with congenital abnormalities and has even
replaced karyotyping for certain reasons for referral. Array CGH provides
valuable information for phenotype-genotype correlation, as well as more
accurate information regarding the clinical significance and the risk in the
current and future pregnancy of the respective patient. Another critical factor
for accurate Copy Number Variation (CNV) classification is parental testing to
determine between familial and de novo CNVs. Appropriate pre and post- test
genetic counceling offer the prospective parents tools to decide on the
management of their pregnancy. However, one of the problems posing
dilemmas to genetic councelors is the fact that it can detect coincidental
findings, variants of unknown significance as well as variants with variable
expressivity.
Array CGH could potentially be used in POC/intrauterine death/stillbirths
samples where malformations exist in the fetuses, using the same platform as

Preface

xi

in prenatal cases, as it offers an increase in detection rate in this category of


samples. Furthermore, this method has the advantage to circumvent technical
problems associated with tissue culturing which may result in failure of
providing results by classical cytogenetics.
In prenatal diagnosis chromosomal analysis is still the primary choice of
testing; for array CGH to replace it, it has to overcome some of its limitations,
such as the detection of CNVs, coincidental or of unclear significance
findings. The detection rate in clinically significant copy number changes
increases with the application of array CGH in cases where the karyotype is
normal and there are sonographic malformations in the fetus. It therefore may
be used more extensively in prenatal diagnosis as well.
Currently there is a lot of debate whether array CGH should also be
introduced in the routine prenatal setting. However, there are still many
technical challenges to be overcome such as:

the resolution
the type of platform to be used
ethical issues including coincidental and of unclear significance
findings

A shared database specifically dedicated to prenatal diagnosis coupled


with the growing amount of data regarding CNVs and dosage sensitive genes
could make it easier to interpret genomic arrays. Currently international
consensus guidelines for the use of array CGH in prenatal diagnosis are still
missing.

In: Prenatal and Maternal Diagnosis


ISBN: 978-1-63482-794-2
Editor: Aaron Henderson
2015 Nova Science Publishers, Inc.

Chapter 1

MATERNAL SMOKING IN PREGNANCY


AND ITS IMPACT ON PLACENTAL AND FETAL
DEVELOPMENT
Emilio Gonzlez-Jimnez, Professor
Department of Nursing. University of Granada, Spain

The consumption of snuff is one of the major public health problems in


the world, and in economically developed countries [1]. Today, Spain is in
phase III of the theoretical model of the tobacco epidemic, characterized by
increased consumption in women, a significant decrease in males and
increased attributable mortality in both sexes even higher among men [2].
Snuff smoke contains thousands of chemicals, specifically over 4,000
different components most harmful to health [3]. The most studied
components are nicotine, tar and carbon monoxide resulting combustion
carbon cigarette. Nicotine is an alkaloid present in the leaf snuff with great
addictive power and speed given its ability to cross the blood brain barrier [4].
In recent years, there has been a reduction in the overall prevalence of
smoking; however, an increased consumption of snuff among younger women,
ie in the reproductive age groups [5] is observed. In connection with the
consumption of snuff during pregnancy, in Spain, there is a high consumption
of snuff during pregnancy [6]. Spanish population studies show a prevalence
of smoking among pregnant women ranges from 22% to 32% [7-10].
However, despite the consumption of snuff is a risk to the health of the fetus,
not all pregnant women manage to leave the snuff during pregnancy [5].

Emilio Gonzlez-Jimnez

Snuff consumption during pregnancy has many adverse effects on the


fetus, ie, spontaneous abortion, premature delivery, placenta previa, premature
sagging of the placenta, vaginal bleeding, high blood pressure and heart rate,
premature rupture of membranes, among others [11].
Among the causes for the increased number of spontaneous abortions in
women smokers include placental malformations, mainly villous infarcts
associated with a deficit of oxygenation and placental blood flow [12]. In
addition, smoking can cause a deficiency in vitamin absorption B once
hydrocyanic acid in cigarettes reduces the levels of this vitamin increases the
risk of preterm birth by 10-20% [13]. Relative to premature rupture of
membranes, the mean relative risk of preterm birth for this cause doubles
among women smokers versus nonsmokers [14], being directly proportional to
the number of cigarettes smoked risk. In addition, these pregnant women who
smoke often have a greater number of infections in the amniotic fluid situation
justifies further cases of polyhydramnios in these pregnant women [11]. For
elevations of blood pressure and heart rate, nicotine is its charge, stimulating
the central nervous system and releasing catecholamines from the adrenal
medulla [15].

EFFECTS OF MATERNAL CONSUMPTION OF SNUFF


ON THE PLACENTA
The placenta is an organ that develops only during the first 3 weeks of
gestation, understanding the processes of preimplantation, implantation and
decidualization, which prepare the body for the differentiation of embryonic
membrane and begin with the formation of the placental membranes [16]. Its
main function is to transport oxygen and nutrients from the maternal blood
into the fetal circulation [17]. Another essential function of the placenta is the
transfer of toxins derived from fetal metabolism toward maternal bloodstream
to be excreted by renal or hepatic pathway [16]. Although women smokers
placenta takes on greater development or hypertrophy as a compensatory
mechanism, it fails to fully meet the nutritional needs of the fetus circumstance
will condition a lower fetal development. In addition, the placenta of women
smokers is ripened early due to the high calcification and subcorinica fibrin
deposition [17]. While in smoking women, the most relevant for their effects
on fetal development placental changes are thickening of the basement
membrane of the trophoblast and reducing the number and light of fetal

Maternal Smoking in Pregnancy and Its Impact on Placental

capillaries [18]. Regarding abnormal placental implantation, snuff accelerates


the development of sclerotic lesions in the tunica media of arteries and
arterioles uterine, causing a reduced blood flow in many areas of the
endometrium [19]. All these changes alter the physiological adaptations
required between mother and fetus, explaining, among other minor issues
anthropometric measurements of fetuses of pregnant women smokers [20].

EFFECTS OF MATERNAL CONSUMPTION OF SNUFF


ON THE FETUS
Different studies confirm that maternal consumption of snuff during
pregnancy can cause serious complications such as low birth weight and
stature, accelerated lung maturation, perinatal mortality, prematurity and
malformations [21]. Thus, it is estimated that maternal consumption of snuff
during pregnancy is responsible for 18% of cases of low birth weight (< 2500
g) and an increased risk of sudden infant death [22, 23]. In relation to low birth
weight is estimated that consumption of snuff in pregnant causes a mean
reduction in birth weight of about 200 grams [22]. The fetal stature also often
affected, falling between 1.2 and 1.3 cm and the cephalic and thoracic
perimeter [24]. Concerning the fetal respiratory system, smoking in pregnant
women alters endocrine mechanisms involved in causing lung maturation
accelerated lung maturation. Also, exposure of the fetus to nicotine is
associated with different morphological abnormalities in the lungs as they are
tortuous and dilated bronchi, decrease in the weight of fetal lung, decreased
fetal lung volume and the number and size of the alveoli [25, 26].
Furthermore, snuff maternal consumption during pregnancy is associated with
an increased risk of fetal death between 28 weeks gestation and 28 days
postpartum life, with a relative risk ranging between 1.2 and 1.6 [27, 28]. It
has also described an association between smoking and increased risk of
preterm delivery. This could be due to vasoconstrictor effect of nicotine in the
placenta attached to the release of catecholamines, effects both with ability to
start your labor [28]. Regarding the development of malformations in fetuses
of pregnant smokers, there is a degree of controversy. Among the main defects
include cleft palate, cleft lip, urogenital malformations, cardiovascular and
hearing and visual impairment [29, 30].

Emilio Gonzlez-Jimnez

CONCLUSION
The available scientific evidence confirms the consequences arising from
the use of snuff during pregnancy maternal health. This circumstance is of
great importance not only for its severity in terms of health but also as the
foundation upon which health policies that promote healthy habits among
pregnant women should be developed. From the diagnosis of pregnancy,
mothers should be adequately informed about the risks for pregnancy and fetus
snuff consumption, making them share in this responsibility and involving
them from the start in the care of their health and fetal wellbeing. Pregnancy,
such as life cycle stage, is the best period to encourage women to abandon the
consumption of snuff. The greater sensitivity of women and close clinical
monitoring can make this period the most suitable for pregnant out of harmful
practices such as smoking. In any case, this high level of involvement between
different health professionals who care for pregnant women to keep women
and their surroundings interest quit this harmful habit is necessary.

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Emilio Gonzlez-Jimnez

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[21] Aguirre Camposano V. Smoking during pregnancy: Effects on
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[27] Elliot J, Vullermin P, Robinson P. Maternal cigarette smoking is
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In: Prenatal and Maternal Diagnosis


ISBN: 978-1-63482-794-2
Editor: Aaron Henderson
2015 Nova Science Publishers, Inc.

Chapter 2

MATERNAL SMOKING DURING PREGNANCY


AND THE RISK OF SLEEP DISTURBANCES
IN INFANTS
Igor A. Kelmanson*
Institute of Special Education and Special Psychology of the Raoul
Wallenberg International University for Family and Child,
St. Petersburg, Russia

ABSTRACT
One of the challenging issues of contemporary pediatrics is potential
long-term effects of maternal cigarette smoking during pregnancy on the
subsequent infant behavioural and cognitive development. Of particular
interest is the question whether, to what extent and in what direction
maternal smoking during pregnancy may influence infant's behaviour
during sleep. This chapter aimed to address the issue. Cigarette smoking
is known to be associated with sleep difficulties in the adults, and several
animal experimental studies suggest that the nicotine contained in
cigarette smoke may interfere with the normal maturation of sleep and
wake. Recent reports were highly indicative that sleep disturbances were
common in the newborns of the mothers who have been heavy smokers
during pregnancy. Changed sleep integrity, sleep disordered breathing
*

Address for correspondence: Professor Igor A. Kelmanson, MD, Ph.D. 92 Bolshaya Ozernaya
Str., St. Petersburg, 194356, Russia. e-mail: iakelmanson@hotmail.com.

Igor A. Kelmanson
and altered arousal mechanisms observed after exposure to tobacco
smoke have been repeatedly implicated to the mechanisms of sudden
infant death syndrome. According to own findings, the babies born to
smoking mothers were more commonly reported as having bedtime
problems and irregular sleep schedule. The mechanisms underlying
changes in sleep due to prenatal tobacco exposure remain to be
elucidated, although it is known that they may be associated with
profound alterations in neurotransmitter disposition, evident in specific
neuronal pathways and persisting after birth.
Conclusion: Maternal smoking during pregnancy is one of the major
predictors of sleep disturbances in infants.

Keywords: infants, sleep disturbances, smoking

According to our own findings based on the study of 200 mother-infant


dyads, 31.5% women smoke during pregnancy. Approximately half of them
smoked both during pregnancy and after infants birth. According to maternal
reports, 62% of pregnancy-smoking mothers smoked less than 10 cigarettes
per day, 32% smoked 10 to 20 cigarettes per day, and 6% smoked more than
20 cigarettes per day. The history of maternal smoking lasted between 5 and
11 years (median 7 years) (I. A. Kelmanson, 2009). Looking at these figures, it
is not surprising that one of the challenging issues of contemporary paediatrics
and perinatology is potential long-term effect(s) of maternal cigarette smoking
during pregnancy on the subsequent infant behavioural and cognitive
development. Specifically, the question is raised whether maternal smoking
may influence infant's behaviour during sleep.
Cigarette smoking is known to be associated with sleep difficulties in the
adults (Soldatos, Kales, Scharf, Bixler, & Kales, 1980), and animal
experimental studies suggest that the nicotine contained in cigarette smoke
may interfere with the normal maturation of sleep and wake (Frank, Srere,
Ledezma, O'Hara, & Heller, 2001). Although maternal smoking in pregnancy
appeared not to alter infant neonatal electroencephalogram (Chernick,
Childiaeva, & Ioffe, 1983) and general infant sleep architecture (Horne,
Franco, Adamson, Groswasser, & Kahn, 2004), some reports were highly
indicative that sleep disturbances were common in the newborns of the
mothers who have been heavy smokers during pregnancy (Pichini & GarciaAlgar, 2006), and that breast-fed infants of the smoking mothers spent
significantly less time sleeping during the hours immediately after their
mothers smoked, compared with the session when their mothers abstained

Maternal Smoking during Pregnancy and the Risk ...

from smoking (Mennella, Yourshaw, & Morgan, 2007). It was found that the
newborns intrauterine exposed to smoking had less optimal Brazelton neonatal
behavioural assessments (Oyemade et al., 1994), abnormal, pitched cry
(Nugent, Lester, Greene, Wieczorek-Deering, & O'Mahony, 1996). These
newborns were more hypertonic and excitable, showed more stress signs and
required more handling than the unexposed ones (Law et al., 2003). Exposed
newborns were more irritable, had more episodes of tremor and sleep
disturbances (Pichini & Garcia-Algar, 2006). It was suggested that the
abstinence mechanism(s) might be responsible for these phenomena; however,
since the adverse effects of nicotine exposure affect multiple transmitter
pathways and influence programming of synaptic competence, the
consequences of early nicotine exposure may appear after periods of apparent
normality (DiFranza, Aligne, & Weitzman, 2004). There is bulk evidence on
the adverse effects of nicotine exposure on foetal and infant development with
obvious nervous system involvement, including altered maturation of visual
evoked potentials (Scher et al., 1998), language and intellectual impairments
(Olds, Henderson, & Tatelbaum, 1994; Tomblin, Hammer, & Zhang, 1998),
colic-like behaviour (Haggart & Giblin, 1988), hypertonicity and increased
nervous system excitation at one month of age (Fried, Watkinson, Dillon, &
Dulberg, 1987), impaired babbling ability at eight months (Obel, Henriksen,
Hedegaard, Secher, & Ostergaard, 1998).

Figure 1. Frequency (percent cases) of the reported sleep problems in infants


depending on maternal smoking habit during pregnancy (I. A. Kelmanson, 2009).

The infants born to smoking mothers were more commonly reported as


having bedtime problems and irregular sleep (I. A. Kelmanson, 2009) (Figure

10

Igor A. Kelmanson

1). Unadjusted odds ratio on infant bedtime problems associated with maternal
smoking during pregnancy was equal to 2.75 (95% CI: 1.15-6.58), and on
irregular sleep was equal to 7.11 (95% CI: 1.24 52.63). Maternal smoking
during pregnancy was found to be one of the major predictors of sleep
disturbances in infancy (I. A. Kelmanson, 2011).
It is now known that in utero exposure to nicotine ensures a harmful effect
on foetal brain development (Ekblad, Korkeila, & Lehtonen, 2015). Tobacco
smoke contains thousands of health-threatening chemicals (Hoffmann &
Hoffmann, 1997), and many of these ingredients are potentially toxic to foetal
development. The major components in tobacco smoke that have been shown
to interfere with foetal brain development are nicotine and carbon monoxide
(Dempsey & Benowitz, 2001). Direct toxic effects from ammonia, polycyclic
aromatic hydrocarbons, hydrogen cyanide, vinyl chloride, and nitrogen oxide
have also been described (Tuthill, Stewart, Coles, Andrews, & Cartlidge,
1999). Nicotine has been shown to cross the placenta, enter the foetal
circulation and accumulate in the foetal compartments from as early as
7 weeks of gestation, in both active and passive smokers (Jauniaux, Gulbis,
Acharya, Thiry, & Rodeck, 1999). Supposedly, nicotine may provoke
teratologic effect on neuronal development in the brain in experimental
animals (Slotkin, Cho, & Whitmore, 1987), and has been shown to affect brain
cell replication and differentiation, leading to changes in brain structure, such
as impaired growth of the forebrain in experimental animals (Chen, Parnell, &
West, 1998). Infants exposed to prenatal smoking have been shown to display
reduced head growth compared with the unexposed ones (Ekblad, et al., 2015).
Maternal smoking during pregnancy has been associated with alterations in
deoxyribonucleic acid (DNA) methylation and dysregulation of microRNA
expression (Knopik, Maccani, Francazio, & McGeary, 2012). The activity of
genes that are important for normal brain development is regulated by DNA
methylation (Martinowich et al., 2003). It has been suggested that these
epigenetic changes may underlie long-lasting modifications of the
development and plasticity of the brain, resulting in later neurodevelopment
problems (Knopik, et al., 2012; Toledo-Rodriguez et al., 2010).
Prenatal exposure to nicotine results in profound alterations in
neurotransmitter disposition, evident in specific neuronal pathways and
persisting after birth (Slotkin, Cho, et al., 1987; Slotkin, Orband-Miller, &
Queen, 1987). Recent findings suggest that one of the sites of action of
nicotine may be in the mesopontine reticular activating system (RAS),
specifically, on the cholinergic pedunculopontine nucleus (PPN) neurons. One
study found that systemic administration of nicotine, or localized injection of a

Maternal Smoking during Pregnancy and the Risk ...

11

nicotinic receptor agonist into the PPN, led to a dose-dependent decrease in


the amplitude of the auditory evoked potential in the experimental animals
(Mamiya, Buchanan, Wallace, Skinner, & Garcia-Rill, 2005). These results
suggest that nicotine, at least initially, has an inhibitory effect on cholinergic
RAS neurons and may reduce the level of arousal. The nicotinic acetylcholine
receptors (nAChRs) are crucial during fetal brain development, as they
modulate axonal path finding, synapse formation and cell survival (Role &
Berg, 1996). The presence of nAChRs can be detected in the human brain and
spinal cord as early as 4 to 5 weeks of gestation (Hellstrom-Lindahl,
Gorbounova, Seiger, Mousavi, & Nordberg, 1998). There is a complex
interaction between nicotine and the RAS. The activation of nAChRs triggers
acetylcholine neurotransmission postsynaptically and the release of other
catecholaminergic neurotransmitters, such as serotonin, dopamine and
epinephrine, presynaptically (Dani, 2001). All these transmitters are critical for
the proper regulation of the sleep-wake cycle and for the maintenance of the
specific sleep stages. It was shown that prenatal exposure to nicotine may
interfere with normal maturation of sleep and wake in the offsprings (Frank, et
al.,
2001).
Prenatal
nicotine
exposure
directly
suppresses
pontogeniculooccipital spike activity, a factor important in initiating and
maintaining active sleep (Vazquez, Guzman-Marin, Salin-Pascual, & DruckerColin, 1996), and indirectly inhibits sleep-promoting neurons in the
ventrolateral preoptic area (Saint-Mleux et al., 2004). Chronic exposure to
exogenous nicotine leads to a high affinity and further desensitisation of the
nAChR, causing long-term changes in the function of the receptor (Cohen et
al., 2005; Weitzman, Gortmaker, & Sobol, 1992). Newborn mice exposed to
nicotine in doses corresponding to the levels human foetuses sustain during
moderate maternal smoking were found to breathe irregularly and had
impaired arousal responses and catecholamine biosynthesis (Cohen et al.,
2002). The development of the dopaminergic system in the offspring may also
be affected (Pauly & Slotkin, 2008).
Loss of function of the specific subtype of nAChRs has been linked to
poor neonatal outcomes, including unstable breathing in sleep (Kahn et al.,
1994) and impaired arousal (Cohen, et al., 2005; I.A. Kelmanson, 2006).
Arousal from sleep is thought to be a major determinant for termination of
apnea, and therefore arousal deficits have been implicated in the
pathophysiology of the sudden infant death syndrome (SIDS) (Kahn et al.,
2002). However, fewer than 10% of apnoeic episodes will be terminated by a
full-fledged electroencephalographic arousal in infants. Autonomic arousals
are nevertheless quite frequent during the period surrounding the termination

12

Igor A. Kelmanson

of an apnoeic event. Prenatal and postnatal exposures to cigarette smoking are


accompanied by decreased arousability in infants (Franco et al., 1999). It has
been shown that infants who became victims of SIDS not only aroused less
from sleep than control infants but their arousal characteristics were different.
SIDS victims had significantly less complete arousals (cortical arousals), but
more incomplete arousals (subcortical activation) than control infants. The
data are suggestive of incomplete arousal processes in infants who eventually
died. More infants of cigarette smoking mothers than control infants failed to
awake to environmental challenges (Kahn et al., 2006). Thus, autonomic and
catecholamine biosynthesis disturbances may contribute to the many-fold
increase in risk of SIDS. Changes in sleep integrity and altered arousal
mechanisms observed after infants are prenatally exposed to tobacco smoke
products have been repeatedly implicated to the mechanisms of SIDS that
usually occurs during sleep (Anderson, Johnson, & Batal, 2005; Kahn, et al.,
2002), and maternal smoking in pregnancy has long been known as one of the
major risk factors of the sudden infant death syndrome (I.A. Kelmanson,
2006).
It was found that either continuation or discontinuation of maternal
smoking after delivery did not significantly add to the baseline risk of sleep
disturbances in infants associated with maternal smoking during pregnancy (I.
A. Kelmanson, 2009). One possible explanation may be an overwhelming
adverse effect of antenatal tobacco exposure compared with the effects of
postnatal infants passive smoking. Meanwhile, this does not exclude
deleterious influence of maternal smoking after birth, and some studies were
indicative that acute episodes of smoking by lactating mothers could alter
infants' sleep/wake patterning (Mennella, et al., 2007).
With full awareness on the fact that further studies in the field are
required, a conclusion may be drawn that maternal smoking during pregnancy
should be considered as a risk factor of sleep disturbances in infants.

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In: Prenatal and Maternal Diagnosis


ISBN: 978-1-63482-794-2
Editor: Aaron Henderson
2015 Nova Science Publishers, Inc.

Chapter 3

PRENATAL SCREENING AND DIAGNOSIS OF


FACIAL CLEFTS: WHERE ARE WE NOW?
William W. K. To1 and Meliza C. W. Kong2
1

MBBS, M Phil, MD, FRCOG, FHKAM (Obs/Gyn)


Chief-of-Service and Consultant Obstetrician
Department of Obstetrics & Gynaecology
United Christian Hospital, Hong Kong
2
MBChB, MRCOG, FHKAM (Obs/Gyn)
Associate Consultant
Department of Obstetrics & Gynaecology
United Christian Hospital, Hong Kong

ABSTRACT
Orofacial clefts are one of the most common non-syndromic
congenital structural abnormalities. In the past, congenital cleft lip and
palate are often only diagnosed after the baby was born. With
developments in ultrasound technology in recent years, the proportion of
cleft lip/ palate that can be detected antenatally has progressively
increased. The wide-spread use of second-trimester ultrasound screening
for morphological abnormalities in the recent two decades have allowed a
very high proportion of significant cleft lip as well as associated cleft
palate defects to be diagnosed antenatally, though the detection rate for

Corresponding author: William WK To, Email: towkw@ha.org.hk

18

William W. K. To and Meliza C. W. Kong


isolated cleft palate (with intact lips) still remains very low. When cleft
lip is first detected on ultrasound examination, a maternal-fetal medicine
specialist will be responsible for performing further detailed scanning to
evaluate whether the facial cleft is an isolated defect, and whether other
genetic syndromes are likely to be associated with the condition. While
conventionally, two-dimensional scanning has been used for screening of
lip clefts, the development of three/ four-dimensional ultrasound scanning
technology has allowed more precise evaluation of the severity of the
cleft lip and the presence and absence of associated alveolar and palatal
clefts. Various three-dimensional scanning techniques to assess such
defects have been described in the literature, including the reverse face
view, flipped face view, oblique face view and so on, but there is still no
consensus as to the most effective and practical methods. In addition,
there is still controversy as to whether any of these techniques would
have better performance than others. As fetal magnetic resonance
imaging gradually becomes an accessible modality of imaging in modern
obstetrics, it is likely to become an additional tool to assess these defects.
All these imaging techniques should yield useful information for
antenatal counselling of the parents, and for planning postnatal
management of the babies.

INTRODUCTION
Facial clefts are among the most common congenital anomalies, with a
point prevalence of approximately 1:500 to 1:1000 live births [1, 2]. Prenatal
detection and diagnosis has been recognized as useful to facilitate prenatal
counselling, to evaluate genetic risks, and to prepare the parents
psychologically to accept and plan for neonatal surgery after birth. To improve
the evaluation of these defectsparticularly those of the palatethree- and
four-dimensional ultrasound (3D/4D US) has been widely introduced as an
additional tool to complement conventional two-dimensional ultrasound (2D
US). Clinically, it is important to differentiate between the different types of
orofacial clefts due to their implications on fetal prognosis. The genetic risks
are believed to be increased when the alveolus or the palate or both are
involved in the facial cleft [3]. There appear to be more associated
malformations and more karyotype abnormalities associated with these more
complex defects, as many syndromes have clefting as part of their phenotype
[4]. While isolated clefts have low perinatal mortality and morbidity, and
primarily pose functional and aesthetic problems after birth, complicated clefts
are associated with a much poorer prognosis. In addition, children with cleft

Prenatal Screening and Diagnosis of Facial Clefts

19

lip plus cleft palate need to undergo more surgical correction procedures than
those with cleft lip alone, and their follow-up more frequently involves
additional orthodontic and orthophonic treatment [5,6]. Thus, when cleft lip is
found on screening using 2D US, precise information about the anatomy of the
palate is important to deliver adequate counselling to the parents on genetic,
surgical, and functional prognostic aspects. A variety of new 3D techniques
have been described in recent years with a view to visualization of the hard
and soft palate, and the use of magnetic resonance imaging (MRI) has also
been advocated. At present, however, there is no consensus as to the best
means to visualize the palate prenatally. This review aims at outlining these
newly developed techniques and discusses their practical utility and
applications.

PREVALENCE OF OROFACIAL CLEFTS IN


DIFFERENT COUNTRIES
The likelihood of encountering alveolar clefts and palatal clefts with cleft
lip varies in different studies, and probably depends on the population being
studied. There are also wide variations in the reported incidence of associated
structural abnormalities. They depend on whether the figures are largely based
on an obstetric cohort evaluated prenatally, or whether the statistics were
collected from a cleft centre receiving referrals to treat babies with significant
clefts.
Classically, Asian populations are reported to have higher rates of facial
clefts, but recent data casts doubts on this generalization. According to the
World Health Organization report compiled from 57 international registries
that were members of either the International Clearinghouse for Birth defects
Monitoring Systems or the EUROCAT for data between 1993 to 1998, a
substantial global geographic variation in the prevalence of orofacial clefts at
birth was observed, ranging from 3.4 to 22.9 per 10,000 live births for cleft lip
and palate and from 1.3 to 25.5 per 10,000 for cleft palate alone [7]. There
were higher rates of orofacial clefts in parts of Latin America and Asian
countries, and lower rates in Israel, South Africa and southern Europe. Apart
from the association of facial clefts in genetic syndromes or as part of
deformities in babies with multiple congenital malformations, various risk
factors have also been proposed for isolated facial clefts. A population based
epidemiological survey of facial clefts in Taiwan from 2002-2009 showed the

20

William W. K. To and Meliza C. W. Kong

overall prevalence to be 0.1% of all births, in keeping with the usual high rates
in Asian populations. Higher prevalence of cleft lip and palate were observed
in multiple pregnancies, being male for cleft lip + palate, being female for cleft
palate only, gestational age < 37 weeks and birth weight < 1.5 kg [8]. A recent
epidemiological study collecting orofacial cleft diagnoses from all provinces
in Canada from 2002 to 2008 surprisingly found that Canada has one of the
highest orofacial cleft birth rates in the world, with a prevalence of 12.7 per
10,000 live births or approximately 1 in 790 live births [9].

PRENATAL DETECTION RATES FOR THE DIFFERENT


TYPES OF CLEFTS
The detection rates for orofacial clefts by US examination also varies
widely among different studies, and the detection rates further differs
significantly for the different types of clefts. In a survey of the detection rates
from 1999 to 2008 in patients referred to a specialist cleft centre in Glasgow,
the overall detection rate was only 15%, but there was a progressive increase
in detection rate from 11% in 1999 to 50% in 2008. Routine US for anomaly
screening was shown to significantly improve the detection rates compared to
scanning high-risk pregnancies only [10].
In a prospective screening in Norway from 1987 to 2004, a total of 101
fetuses or newborns with facial clefts were found in a population of 49 314
deliveries.
The distribution of clefts was: 25% cleft lip, 51% cleft lip plus cleft palate,
and 24% cleft palate. No cleft palate was detected antenatally. Cleft lip with or
without cleft palate was detected prenatally in 45% of the cases, with a
significant increase in the detection rate from 34% in 1987-1995 to 58% from
1996 to 2004; 69% of all the cases were first detected at routine second
trimester ultrasound examinations; 43% of the cases of cleft lip plus cleft
palate and 58% of those with cleft palate only had associated anomalies.
Overall, 12% of these patients had associated chromosomal aberrations, and in
18% the clefts were part of a syndrome or sequence [11].
In a recent large prospective US screening of 35 000 low-risk and 2800
high-risk pregnant women in the Netherlands, orofacial clefts were detected in
62 fetuses, giving a point prevalence of 1:613. The distribution of
abnormalities was: 29% cleft lip alone, 40% cleft lip plus cleft palate, and 27%
cleft palate only; there was also one rare median cleft and one atypical cleft.

Prenatal Screening and Diagnosis of Facial Clefts

21

Regarding these anomalies, 61% were unilateral, 37% were bilateral, and 39%
had associated abnormalities (chromosomal defects in the cleft lip with cleft
palate group and cleft palate only patients). The sensitivity of detecting cleft
lip with or without cleft palate prenatally was 88%. Cleft palate only was not
detected prenatally in any conceptus. There were three false-positive cases,
two of whom had other multiple abnormalities. It was concluded that in a lowrisk population, US screening to detect cleft lip with or without a palatal cleft
had high sensitivity [12]. In a series of 570 children referred to a facial cleft
centre in the United Kingdom, it was found that the frequency of associated
structural abnormalities varied with the anatomical type of the cleft, being
9.8% for unilateral cleft lip plus palate, 25% for bilateral cleft lip plus palate,
and 100% in those with a midline cleft lip and palate. Of the 252 cases with
isolated cleft palate, 5.6% had either karyotypes or associated structural
abnormalities and 21% had a genetic syndrome as an underlying diagnosis.
However, none of the palatal clefts without facial clefts were identified
antenatally [13]. In a Swedish series including data from 2006 -2010, the
detection rate among all 144 orofacial clefts was only 31%, but if isolated cleft
palate were excluded, the detection rate was slightly better at 43%. Among
those that were prenatally diagnosed, 57% were diagnosed before 20 weeks
gestation, and only 43% were anatomically accurate as compared to postnatal
findings of the babies. The authors concluded that the performance can be
improved by increasing focus on the detection of clefts during scanning,
standardizing scan protocols and rescanning when facial views were
incomplete [14]. On the other hand, a Finnish series including 214 facial clefts
treated between 1998 to 2011at one centre showed that cleft palate was the
most common, accounting for 67% of all cases, followed by cleft lip and
palate (18.7%) and then cleft alveolus (12%). Left sided clefts accounted for
82% while right sided clefts only 18%, and around 20% of the affected babies
would have family history of clefts. The authors concluded that these northern
Finland figures were quite different from that of other European populations,
implying that there could be genetic variances [15].

ACCURACY OF PRENATAL DIAGNOSIS OF THE DIFFERENT


TYPES OF CLEFTS
The accuracy of antenatal ultrasound diagnosis of cleft lip and palate was
studied in a series of 96 cases in an expert French centre with a mean

22

William W. K. To and Meliza C. W. Kong

gestational age at examination of 28 weeks. The sonographic appearance of


cleft lip, cleft lip with cleft alveolus, and cleft lip plus cleft palate was
subsequently confirmed in 88% of the cases. Overestimation of the degree of
cleft occurred in eight cases, and under-estimation occurred in three. Thus, the
authors believed that inclusion of 3D and 4D US imaging allows easier and
more precise evaluation of the different cleft constituents [16]. In another
retrospective review of surviving cases between 2002 and 2003 at a cleft
surgical referral centre in London, of 149 with a cleft lip with or without cleft
palate, 59% were diagnosed based on antenatal ultrasound examination,
though 25% of the latter entailed minor reporting errors. The latter included:
errors in describing the side and type of the lip cleft (12%), predicting the
possibility of cleft palate (12%), and recognizing the anomaly (1%). There
were 102 cases of isolated cleft palate, of which none were detected
antenatally. It was concluded that inaccuracies in antenatal ultrasound reports
occur frequently when attempting to determine the type of cleft lip and when
assessing whether there was a cleft palate [17].
To verify the accuracy of prenatal axial 3D US in predicting the absence
or presence of cleft palate, in the presence of a cleft lip, 79 patients with a
prenatal 2D US screening diagnosis of unilateral or bilateral cleft lip at 22 to
25 weeks were subjected to axial 3D US of the fetal palate. The findings were
compared to those noted at births. It was found that 77 out of 79 prenatal
predictions were correct, yielding a sensitivity and specificity to detect cleft lip
and palate (in this high-risk population) of 100% and 90%, respectively. Thus,
the study confirmed that 3D US of the hard palate showed high accuracy in
identifying prenatal cleft palate when cleft lip is identified at mid-trimester
screening scan [18].
In 124 cases of suspected orofacial clefting diagnosed by routine 2D US,
there were 110 who had isolated facial clefts, and 100 having successful
reverse face views were analyzed. The sensitivity of the 2D enhanced with 3D
reverse view technique for the diagnosis of cleft lip was 95% (false positive
rate, 8%), for alveolar ridge 85% (7%) and for hard palate 90% (16%). It was
concluded that the reverse face view was possible in 90% of fetuses in whom
90% have a correct classification of clefts of the lip, alveolar ridge
and palate [19].
A recent systematic review reveals the diagnostic accuracy of secondtrimester transabdominal ultrasound in detecting orofacial clefts in low- and
high-risk populations and to compare 2D US and 3D US techniques [4]. This
review included 27 studies, of which 21 involved unselected low-risk
populations and six entailed high-risk populations. There was diversity in the

Prenatal Screening and Diagnosis of Facial Clefts

23

gestational age at which the ultrasound examinations were performed, ranging


from 15 to 36 weeks. There were also considerable variations in the diagnostic
accuracy of 2D US in low risk women; prenatal detection rates ranged from 9
to 100% for cleft lip with or without cleft palate, 0 to 22% for cleft palate only,
and 0 to 73% for all types of clefts. Notably, 3D US in high-risk women
resulted in a detection rate of 100% for cleft lip, 86 to 90% for cleft lip with
cleft palate, and 0 to 89% for cleft palate only. The inclusion of older studies
with less advanced ultrasound machines and the varying levels of training and
expertise in individual studies all contributed to the very wide range of
detection rates quoted in this review. However, such variations could be a true
reflection of the very heterogeneous performance in the detection of orofacial
clefts in practice. Thus, 2D US screening for cleft lip and palate in a low-risk
population has a relatively low detection rate, approaching 0% for isolated
cleft palate, but is associated with few false positives. Three-dimensional
ultrasound can achieve a reliable diagnosis, but not for isolated cleft palate [4].

CONVENTIONAL TWO- VERSUS THREE


DIMENSIONAL ULTRASOUND
Conventional 2D examination of the face requires the mid-sagittal plane
and that a series of images in the anterior coronal plane be obtained by probe
manipulation by moving out from the nose and oral cavity to the edge of the
lips so as to obtain the nose-mouth view. Examination of the palate is more
difficult with conventional 2D US. The most common method is to obtain
serial axial/transverse images from the nose down through the oral cavity to
the lower edge of the mandible. By this means, the alveolar ridge, palate,
tongue, and mandible can be visualized consecutively. Sagittal views are
useful for visualizing the facial profile; particularly in bilateral cleft lips a
protrusion of the detached pre-maxillary mass may be visualized [20]. Other
2D US approaches that might increase detection rates include the use of
transvaginal ultrasound early in the second trimester [21] or the addition of
colour flow Doppler [22], though both of these techniques seem to have
become obsolete with the advent of 3D US.
Standard 3D US assessment of the facial profile included the orthogonal
display mode and surface rendering (Figure 1). The orthogonal display mode
allows the simultaneous analysis of the three reference planes: sagittal,
transverse, and coronal.

Figure 1. Three-dimensional examination with three orthogonal plans and surface rendering.

Prenatal Screening and Diagnosis of Facial Clefts

25

Thus, once the presence of a facial cleft is suspected, the three reference
planes are imaged to characterize the anatomical defect. The alveolus and
palate can usually be identified in the transverse plane by visualizing the front
tooth buds and alveolar ridge and then by rotating the volume slightly to
examine the symmetry of the palate. The surface rendering allows imaging of
the soft tissue of the face and its relationship to underlying bony structures.
Currently, standard 2D US is used for routine mid-trimester morphology
scans, screening for cleft lip being an essential part of the protocol for most
centres. A study comparing the trends in prenatal diagnosis of facial clefts
before and after the introduction of the 20 week-fetal anomaly scan in 2007 in
the Netherlands showed that the introduction of routine fetal morphology scan
has decreased the gestational age at diagnosis of cleft lip +/- cleft palate. A
total of 123 cases of orofacial clefs were analyzed of which 76% were
diagnosed before 24 weeks, 46 of which were associated with other structural
abnormalities and 76 were isolated, while one was not confirmed after birth.
The median gestational age at diagnosis decreased by 2 weeks after 2007. The
proportion of isolated facial clefts detected antenatally increased significantly
after 2007. The overall detection rate of orofacial clefts increased from 43%
before 2007 to 86% after 2007 without an associated increase in terminations
of pregnancies [24].
When a cleft lip is diagnosed, efforts are then made to evaluate the extent
of the lesion and the presence or absence of associated alveolar and palatal
clefts. Rendered 3D images may also provide more-easy-to-understand
landmarks for the planar views, and at the same time facilitate counseling of
the family and a consultation with a surgeon to explain the abnormality [23].
Thus, at present, 3D/4D imaging is most commonly employed for such
secondary evaluation and joint counselling. Recourse to 3D/4D scanning as a
primary tool for screening of facial clefts could be time-consuming and has not
been shown to be cost-effective. The use of rendered images alone has been
reported to introduce false positives due to the appearance of pseudo-clefts
that are usually due to rendering artefacts or acoustic shadows, which lead to a
loss of specificity for the ultrasound diagnosis [25, 26].
The widespread use of 3D US for assessment of facial clefts also led to
issues of training in 3D/4D scanning. Inexperienced sonographers may find
the learning curve for volume reconstruction very time consuming and
unrewarding. In an interesting study on such training, using a standardized 3D
US protocol to acquire 260 volumes, operators with different levels of
competence were tested on their performance of post-processing 3D face
volumes to detect facial clefts, the ability to reconstruct the facial volumes,

26

William W. K. To and Meliza C. W. Kong

and the time needed to reconstruct each plane to allow proper diagnosis of a
cleft. It was found that the three orthogonal planes of the fetal face were
adequately reconstructed with similar performance when acquired by a
maternal fetal medicine specialist or by residents with minimal experience.
The learning curve for manipulation of 3D US volumes of the fetal face
corresponds to 30 cases only and is independent of the operators scanning
experience. It was concluded that inexperienced sonographers can successfully
visualize and assess a 3D image volume of the fetal face using such standard
protocol [27].

The Equals Sign


Notwithstanding the emphasis on 3D US techniques, a novel marker for
the diagnosis of isolated fetal cleft palate using 2D US has been recently
described and is termed the equals sign [28]. This uses a transverse plane to
visualize the uvula and epiglottis as well as a mid-sagittal pane to visualize the
soft palate and the uvula. In this German series of 667 consecutive women
examined between 20 and 25 weeks, a normal uvula could be visualized with
the typical echo pattern (equals sign) in 90.7% of cases and the soft palate
could be completely visualized in the median sagittal section in 85.3%.
However, the number of actual abnormalities included in this series was small,
and only one case of isolated cleft palate and one case of cleft lip and palate
were correctly diagnosed [28]. Further clinical experience with this technique
for routine use is required to evaluate its sensitivity and specificity for the
detection of isolated palatal clefts.

New Techniques with Three-Dimensional Ultrasound


A new 3D US technique called the 3D reverse view was first described by
Campbell et al. in 2005 [29]. The case series described eight consecutive cases
of suspected orofacial clefting that were examined by 3D surface rendering.
The fetal lips and alveolar ridge were examined in the frontal plane and the
face was then rotated through 180 degrees on the vertical axis to examine the
secondary palate by 3D reverse face view. The investigators concluded that the
3D reverse face technique allowed relatively straightforward assessment of the
fetal palate with a high degree of accuracy.

Prenatal Screening and Diagnosis of Facial Clefts

27

Platt et al. [30] then described the flipped face view in 2006. The fetal face
was initially examined with the fetus in the supine position, and using 3D US,
a static volume was acquired. The acquired volume was then rotated 90
degrees so that the cut plane was directed in a plane from the chin to the nose.
The volume cut plane was then scrolled from the chin to the nose to examine
the lower lip, mandible, alveolar ridge, tongue, upper lip, maxilla and alveolar
ridge, and hard and soft palates, in sequential order. The authors promoted the
practicality of this approach to identify the full length and width of the
structures of the mouth and palate, which allowed the examiner to identify
normal anatomy as well as the clefts of hard and soft palates. The oblique face
view was described by Pilu and Segata [31] in 2007 to visualize the secondary
palate. To avoid acoustic shadowing from the alveolar ridge, the secondary
palate was insonated at a 45-degree angle in the sagittal plane and 3D US was
used to reconstruct axial and coronal planes. In this small series, the secondary
palate was successfully visualized in 10 of 15 fetuses, both in the axial and
coronal planes. In the only fetus in this series with cleft lip, the palatal lesion
was clearly demonstrated in the coronal plane [25] In a study to compare the
performance of the reverse face, flipped face, and oblique face methods for
visualization of the hard and soft palate, a total of 60 fetuses (10 of which had
facial clefts) with a gestation of 23 to 33 weeks were examined, with the result
that the upper lip and alveolar ridge were well visualized in all cases with the
three methods. Involvement of the hard palate was diagnosed accurately in
71% with the reverse face view, in 86% with the flipped face view, and in
100% with the oblique face view. The hard palate was correctly found to be
normal in 78%, 84% and 86% of the 50 normal fetuses, respectively.
Involvement of the soft palate was diagnosed correctly in only one of seven
fetuses with secondary palate defects in the flipped face and oblique face
views, and was correctly considered to be intact in only 16% and 26% of
normal fetuses using these two views, respectively.
It was concluded that the oblique or flipped face views make it possible to
visualize the soft palate in selected cases [32]. Actual visualization of the soft
palate requires an excellent initially acquired volume, fluid between the tongue
and the palate, and a curving plane to follow the structure of the palate;
evidently this was not possible with the reverse face view (Figure 2).
In a retrospective review of 42 cases in predicting the presence or absence
of associated alveolar clefts / palate clefts in the presence of lip clefts using
available 3D techniques, there were 5 cases where prenatal USG overdiagnosed the severity of the cleft, while 3 cases had underdiagnoses, giving
an overall accuracy of 80% (34/42).

28

William W. K. To and Meliza C. W. Kong

Figure 2. Facial cleft as visualised by two-dimensional (2D) ultrasound (US) and 3D


rendering. (a) Cleft lip as visualised by 2D US; (b) cleft lip as visualised by 3D US; (c)
cleft palate seen in coronal plane reverse face view; (d) cleft alveolar and palate in
axial plane visualised by flipped face view; and (e) cleft alveolar and palate in axial
plane as visualized by oblique face view.

The most common discrepancy was in the over- or under diagnosis of


alveolar clefts, while there were no errors concerning the side of the cleft.
When only the prenatal diagnostic accuracy of presence or absence of cleft
palate was calculated, the overall accuracy was 95% (40/42, association 0.90).
It was concluded that the prediction of presence or absence of palate clefts in
the presence of lip clefts was good, but the precise prediction of alveolar clefts
was most prone to errors. The limitations of such USG predictions should be
explained to the mothers at the time of counselling (Figure 3) [33].
Efforts were also made to detect orofacial clefts in the first trimester. In a
study of 237 fetuses undergoing first trimester US screening for aneuploidy,
3D datasets were collected and analyzed offline to evaluate the primary palate

Prenatal Screening and Diagnosis of Facial Clefts

29

in the coronal plane at the base of the retronasal triangle, and the secondary
palate by virtual navigation in the axial plane.

A. Facial cleft as seen by 3D USG.


B. Lip cleft with intact alveolar ridge.
C. Associated alveolar cleft as visualized by rotational 3D views.
D. Associated palate cleft as visualized by rotational 3D views.
Figure 3. Lip cleft, alveolar cleft and palate cleft as visualized by 3D USG surface
rendered images.

The quality of these first trimester 3D datasets were classified as good,


fair or poor in 76%, 20% and 4% respectively. Of the 9 fetuses suspected to
have a cleft of the primary palate, 7 were confirmed after birth or at
postmortem after abortion. The false positive was only 0.9% Of the 6 cases
suspected to have secondary palate cleft, all were confirmed, but 1 case was
missed and was diagnosed subsequently at 16 weeks. The authors concluded
that all cases of primary palate clefting and 86% of secondary palate clefts
were visualized using 3D US with a satisfactory false positive rate in the first

30

William W. K. To and Meliza C. W. Kong

trimester [34]. These results are indeed excellent and much better than many
other series auditing second trimester screening. However, it remains doubtful
whether such good results can be achieved in an average centre. In fact, in
another recent study, the secondary palate was assessed in 45 fetuses with
normal face anatomy and 4 fetuses with malformations (including retrognathia
and/r micrognathia, cleft lip +/- cleft primary palate) between 12 to 16 weeks
gestation. The secondary palate was visualized only in 19/49 (38%) of the
fetuses and in 2/49, only the hard palate, in 6/49 only soft palate. Both soft and
hard palate were demonstrable only in 11/49 [35].

Other Three-Dimensional Ultrasound Techniques


In a series of 100 fetuses at advancing gestations from 17 weeks to 32
weeks, US scans were performed using the strict anterior axial plane of the
reconstruction volume and the underside 3D view of the fetal palate. This 3D
view of the fetal palate was then compared with the normal anatomical view of
the fetal palate obtained by surgical fetopathological examination of the palate
for fetuses of corresponding gestation. Three-dimensional imaging of the fetal
maxilla and secondary palate was possible and the overall reliability of
visualization across the gestational ages was medium to very high (0.73 to
0.96). It was concluded that this technique of anterior axial 3D view
reconstruction of the fetal palate seen by an underside view can provide useful
information of the integrity of the secondary palate [36].
Using acquired routine 3D volume, a technique to obtain a sweeping view
of the fetal soft and hard palates has been reported. The secondary palate was
viewed in three oblique planes targeted at the uvula: the oblique axial, the
oblique sagittal, and the reverse face view in 31 normal fetuses between 15
and 35 weeks. It was found that the various surfaces of the secondary palate
could be viewed in all fetuses in the oblique axial and the reverse face views,
and in all except two fetuses of less than 19 weeks in the oblique sagittal view.
Thus, rotating or tilting of these orthogonal planar images of the fetal head
allowed the visualization of the various aspects of the palate in most fetuses,
with the uvular as a useful landmark. However, due to the relatively small size
of the uvula, its demarcation at an early gestation before 20 weeks is
not easy [37].
Three-dimensional extended imaging (3DTI; Accuvix, Medison Co Ltd,
Seoul, Korea) has been described since 2005 as a new modality to examine
various fetal structures. In a pilot study, Leung et al. [38] reported the results

Prenatal Screening and Diagnosis of Facial Clefts

31

in examining six cases of facial clefts. The method was found to be


advantageous over 2D US in at least one of the cases by allowing
simultaneous display of bilateral cleft lip and palate which were located in two
different axial plans [38]. However, the diagnostic accuracy for facial cleft
between 2D US and 3D US was not directly compared.
In a subsequent study by the same group [39], fetuses suspected of having
a facial cleft by previous ultrasound examination or family history were
examined with 2D and then 3D US. A total of 30 cases were analyzed to
compare the performance of 2D US and 3DTI, of which 22 had cleft lip and
nine also had cleft palate at birth. The use of 2D US with or without 3D US
correctly identified all cases of cleft lips prenatally. However, the use of 2D
US in conjunction with 3D US correctly identified more palatal clefts than 2D
US alone (89% vs 22%, P<0.01). Primary palatal cleft was well-demonstrated
in both multi-slice view (MSV; Accuvix, Medison Co Ltd, Seoul, Korea) and
orthogonal display modes. There were no false positives as all unaffected
fetuses were reported as having no cleft palate with the use of the MSV mode.
The combined approach using 2D US and 3D US with the 3DTI techniques
offering both orthogonal display and MSV modes significantly improved the
prenatal detection rate for cleft palate compared to 2D US alone, without any
decrease in specificity.
Recently, the use of a novel reslicing technique to examine the retronasal
triangle of fetuses in the first trimester was studied. The authors obtained 3D
volume data sets from 100 low risk and 50 high risk first trimester fetuses, and
was successful in evaluating offline the retronasal triangle in 96% of the cases.
The secondary palate could be assessed in 93% and an abnormal retronasal
triangle in the coronal plane was detected in 2 cases of lethal aneuploidy. The
overall false positive rate was 1.33%. The authors concluded that abnormal
retronasal triangle was apparently a valuable marker for early diagnosis of
facial clefting. The new reslicing technique was proven to be easy, fast and
accurate and suggested that the technique could be included in daily
practice [40].
It has to be noted, however, that the studies quoted in these two sections
were primarily concerned with the methodology and the sonographic approach
used to provide images of the palatal structures in largely normal fetuses,
whilst the number of pathologies described was relatively small. Faure et al.
[36] and Wong et al. [37] included no abnormal cases at all, Platt et al. [30]
and Pilu and Segata [31] showed one abnormal case as an example, Tonni et
al. [40] described only 2 abnormal cases, Campbell et al. [29] and Ten et al.
[32] described eight and 10 cases respectively, and Wang et al. [39] described

32

William W. K. To and Meliza C. W. Kong

22 cases. In addition, many of these series were retrospective comparisons to


test the US technique under study instead of being prospective studies to
assess the sensitivity or specify of these techniques. Moreover, while 3D US
techniques appeared to improve the diagnosis of palatal cleft with associated
cleft lip, as with studies on 2D US, none of the 3D studies described the
detection of an isolated cleft palate. Further large prospective trials to compare
the efficacy and accuracy of these techniques are obviously needed.

Fetal Magnetic Resonance Imaging


The role of fetal magnetic resonance imaging (MRI) was evaluated in
various studies to assess whether it should be complementary to US in the
evaluation of cleft lip and cleft palate. In one such series, to assess whether the
orofacial cleft was isolated or in association with syndromic conditions, 27
fetuses with US-diagnosed cleft lip or cleft lip/palate were recruited to
undergo fetal MRI examination. Their facial skeleton, central nervous system,
and fetal body was studied at a mean gestational age of 24 weeks. The
diagnosis of cleft lip/palate was confirmed in 16 of 25 fetuses, and additional
information about the extent of the cleft and the degree of involvement of the
anterior and posterior palate was obtained in eight of these fetuses. The MRI
ruled out the diagnosis in one of the 25 fetuses. It was concluded that MRI was
able to define the degree of involvement of the posterior palate and the lateral
extent of the cleft with higher diagnostic accuracy than US. Furthermore, MRI
provides a complete study of the fetal head and biometric development of the
facial bones, thus enabling early detection of potential syndromic conditions
[41]. To assess whether the use of fetal MRI could provide a definitive
prenatal diagnosis of cleft palate, 49 pregnant women with fetuses with
diagnosed facial clefts from routine 20-week morphology scans were
subjected to fetal MRI at between 24 and 37 weeks. The positive predictive
value of fetal MRI for involvement of the palate was 96%, and the negative
predictive value was 80%, even when the radiologist was blinded to the US
findings. The accuracy of the radiologist in predicting palatal clefting from
different MRI signs improved significantly over a short learning curve. Thus,
fetal MRI enabled more accurate prediction of the extent of a cleft palate after
ultrasound diagnosis of a cleft lip [42].
In a retrospective review of 60 brain-targeted MRI of fetuses from 20 to
38 weeks gestation, in which 55 were normal and 5 had orofacial clefts, 2
independent radiologists were able to demonstrate a high degree of

Prenatal Screening and Diagnosis of Facial Clefts

33

interobserver agreement. Normality was correctly scored in 96% to 100% of


normal lips and primary palates and in 93% and 97% of normal secondary
palates, and all pathological cases were identified. The authors concluded that
brain-targeted fetal MRI in experienced hands were apparently highly accurate
for evaluation of orofacial cleft beyond 20 weeks, though assessment of the
secondary palate might be slightly more limited than the lip or
primary palate [43].
In a series of 34 Austrian women with a mean gestation of 26 weeks
(range, 19-34 weeks), in-utero MRI performed after ultrasound examination
had identified either a facial cleft and/or other structural abnormalities. In all,
32% of the cases had primary palatal clefts alone, 59% had clefts of the
secondary palate, and three had isolated clefts of the secondary palate. In all
cases, the primary and secondary palate were visualized successfully with
MRI. Compared to ultrasound that detect 15% of the facial clefts and
misclassified 44% of them, the MRI classification correlated well with the
postnatal or postmortem diagnosis. It was concluded that MRI allowed
detailed prenatal evaluation of the primary and secondary palate, and that by
demonstrating palatal involvement, it provided a better detection rate and
classification of facial clefts than ultrasound alone [44].
In another retrospective series of 62 cases where US had either suspected
a facial cleft, or where there were difficulties with visualization by US,
additional MRI examination between 22-35 weeks gestation was able to show
normal fetal facial features to confirm normality in 47 cases, to confirm the
suspected diagnosis of orofacial clefts in 10 other cases (66.6% of the 15 with
orofacial clefts), and to reveal more details of the facial clefts in 5 cases
(33.3%). MRI examination also yielded additional information apart from the
facial cleft in 10 of these 15 cases, including showing various central nervous
system or renal o abnormalities [44]. The authors concluded that prenatal MRI
should be a method supporting US, which could confirm or expand US
diagnosis, but could also change it. In the presence of cleft lip and palate, fetal
MRI produced a better picture of the connections between the cavities, the
degree of involvement of the secondary palate and cleft extent, and also helped
to detect and assess other associated fetal abnormalities [45].
It must be noted, however, that the data on MRI studies of orofacial clefts
quoted above were performed beyond 20 weeks, mostly between 24 to 37
weeks of gestation, and the utility of earlier MRIs (around 20 weeks of
gestation) when ultrasound diagnoses were usually made remained untested.
Further studies are needed to compare the performance of ultrasound and MRI
at equivalent gestation ages. The use of MRI findings for prenatal diagnosis

34

William W. K. To and Meliza C. W. Kong

and clinical counselling to parents targeted at decisions for terminating or


continuation of the pregnancy remains limited at present.

Prenatal Diagnosis Leading to Prenatal Surgery?


Recent developments in video-endoscopic technology have boosted the
development of operative techniques for feto-endoscopic surgery, which has
been demonstrated to be less invasive than the open approach. Experimental
intrauterine correction of cleft lip and palate has been recently performed
using such an approach. The main advantages of prenatal surgery for this non
life threatening condition included scarless fetal wound healing and bone
healing, enabling better or normal maxillary bone growth and better cosmesis
[46] by correcting the primary deformity. Scarless fetal lip and palate repairs
may prevent the ripple effect of postnatal scarring with its resultant secondary
dentoalveolar and mid-face growth deformities, and might dramatically reduce
the number of postnatal reconstructive procedures that these children might
need to undergo [47]. Nevertheless, such advantages of prenatal surgery
remain theoretical, and clinical data on its effects are still not yet available in
the literature.

CONCLUSION
While there were wide variations in the reported prenatal detection rates of
facial clefts, it is evident that the performance of 2D US screening has
progressively improved in recent years. While 3D US screening alone should
probably not be used due to the higher false-positive rates, the development of
3D US techniques has improved the detection of palatal clefts associated with
cleft lip, by allowing direct visualization of at least part of the secondary
palate.
On the other hand, the performance, precision and clinical practicality of
these new techniques at different gestational ages have not been thoroughly
compared or evaluated. More experience in learning and applying these
techniques needs to be accumulated. Isolated palatal clefts remain problematic,
and detection rates remain low even with the addition of 3D US. The use of
MRI to facilitate prenatal assessment of facial clefts should be further
developed, with the increasing availability of fetal MRI in many centres.
Prenatal surgery for congenital clefts remains a goal for the future.

Prenatal Screening and Diagnosis of Facial Clefts

35

(Part of the materials of this chapter was adopted from a review article by
the first author. To W WK. Prenatal diagnosis and assessment of facial clefts:
where are we now? Hong Kong Med J 2012; 18: 146-152)

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In: Prenatal and Maternal Diagnosis


ISBN: 978-1-63482-794-2
Editor: Aaron Henderson
2015 Nova Science Publishers, Inc.

Chapter 4

NONINVASIVE PRENATAL DIAGNOSIS BY


SEQUENCING CIRCULATING FETAL DNA
Liang Xu1 and Rui Shi2
1

Research Center for Translational Medicine, Shanghai East Hospital,


Tongji University, Shanghai, China
2
Department of Obstetrics and Gynecology, Shanghai East Hospital,
Tongji University School of Medicine, Shanghai, China

ABSTRACT
Nowadays, prenatal diagnosis is necessary for pregnant women. For
the parents who are expecting a child, the genetic test may provide the
information whether they are carrying rare gene mutations and whether
they are at risk of passing them onto their offspring. However, the
ultimate determination of genetic diseases often requires invasive
procedures such as amniocentesis and chorionic villus sampling, which
may cause fetal miscarriage. A noninvasive type of prenatal diagnosis
needs to be developed in clinical practice to dispel safety concerns. In this
paper we will introduce the technical advancement of using maternal
circulating nucleic acids as the sample in noninvasive studies, and
highlight the utilization of next-generation sequencing in the screening of
genetic diseases.

Correspondence: Rui Shi, M.D. Department of Obstetrics and Gynecology, Shanghai East
Hospital, Tongji University School of Medicine, No.150, Ji Mo Road, Shanghai 200120,
China. Tel: +86-21-58766224. Fax: +86-21-58766224. Email: shezzle@126.com

42

Liang Xu and Rui Shi

Keywords: noninvasive; prenatal diagnosis; next-generation sequencing;


circulating fetal DNA

1. INTRODUCTION
There are more than 3000 Mendelian disorders [1] accounting for about
20% of deaths in infancy [2]. Besides the routine ultrasound examination
concerning fetal development, for those who are at high risk of genetic
disease, prenatal diagnosis usually consists of diagnosis of chromosomal
aneuploidies and several single-gene disorders. Current prenatal screening of
pregnant women considers only a few specific diseases such as trisomy 21, the
genetic cause of Down syndrome. Although there are many noninvasive
screening approaches available, an invasive approach is still the gold standard
for prenatal diagnosis of genetic disorders. The risk of complications related to
abortion is as high as 0.5% [3, 4], and the miscarriage rate after chorionic
villus sampling is as high as 6.8% [5]. An ideal prenatal genetic diagnostic
screening should be precise and noninvasive to meet the clinical demand.

2. CHARACTERISTICS OF CIRCULATING FETAL DNA


In the plasma of pregnant women, DNA molecules are mainly short DNA
fragments. The DNA size in pregnant women is significantly larger than that
in nonpregnant women [6]. Since the evidence by Lo et al. [7] in 1997 that
cell-free fetal DNA exists in maternal plasma, much of fetal DNA nature has
been uncovered. Fetal DNA can be traced in maternal plasma as early as day
18 after the embryo transfer [8]. As gestation progresses, the amount of fetal
DNA increases, and usually reaches 3-6% of total cell-free DNA on average
[7].
The origin of this fetal DNA, as many documents indicate, derives from
the maternal placenta [9, 10]. The apoptosis and necrosis of placental cells
contribute to fetal DNA release. Fetal DNA molecules are shorter than
maternal ones, most of which are less than 300 bp [6]. The circulating fetal
DNA cannot persist for a long time, for its concentration dramatically
decreases postpartum [11-13].

Non-Invasive Prenatal Diagnosis ...

43

3. DIAGNOSIS USING CIRCULATING FETAL


NUCLEIC ACIDS
As the content of fetal nucleic acids can reach 13% of total DNA in
maternal cell-free plasma [14], this gives the opportunity to detect information
about the fetal genome. The simplest idea is to examine sex-linked diseases.
Congenital adrenal hyperplasia, for instance, is a problem of virilization of the
genitalia related only to female fetuses [15]. If genes specifically residing in Y
chromosome sequences are detected in maternal circulating DNA, the fetus is
not at risk of suffering this disorder [16]. Theoretically, any Y chromosomespecific sequence such as Sox14 or Tbx3 [14] could be a sex determination
marker in clinical detection, and generally it can reach 95.4% sensitivity and
98.6% specificity [17]. Similarly, the ones that do not exist in the mother's
genome but are carried by the fetus can also be used for analyzing genetic
disease. Rhesus D (RHD) belongs to this category. It encodes rhesus blood
group antigen D in RHD-positive fetus and will induce hemolysis of RHD
incompatibility in RHD-negative mothers. Such qualitative assessments of
maternal and fetal DNA in a specific genome region are convenient and
reliable. The procedure of standard PCR is easy to perform, the accuracy is
satisfactory (close to 100%) [18], and adapted to the analysis of other fetal
genetic disorders such as myotonic dystrophy and -thalassemia [19-21].
Although previous studies have shown that quantitative analysis of mRNA in
maternal plasma using RT-PCR could monitor disease condition and progress
[22-24], or even detect chromosome 21-encoded mRNA [25], it is not feasible
and applicable in clinics, because it is difficult to make a determined value (or
relative multiples) to define a disease.
PCR-based qualitative analysis can only define a small portion of genetic
disorders. More single-gene disorders often involve gene polymorphisms or
mutations, so the screening of genetic diseases demands the detection to the
extent of the single base level, which requires improvement of detection
technology and combination of multiple tools. This has been achieved by
Dennis Lo's group. They detected -thalassemia mutations using single-allele
base extension reaction and mass spectrometry (MS) [26]. In this method, a set
of well-designed primers is applied to specifically amplify mutant fetal allele,
but not to the maternal allele (father is the carrier of the mutation), so the
following mass spectrometry assay is able to easily identify the presence of a
mutant fetal allele. The PCR-MS strategy is also applicable to detect fetal

44

Liang Xu and Rui Shi

trisomy 21 by determining the ratio between single nucleotide polymorphism


(SNP) alleles in PLAC4 mRNA [27].

4. SEQUENCING CIRCULATING FETAL DNA


PCR assay combined with MS does permits the chance of gene
polymorphism detection, but it seems difficult to cover multiple genes or SNP
alleles, so researchers attempt to directly sequence the maternal plasma DNA
in order to acquire multiplex detection of the fetal genome noninvasively.
Fetal DNA represents a small fraction in maternal plasma compared to
predominant maternal DNA, therefore the sequencing detection should be
sensitive enough to capture the dim fetal DNA. By means of massively
parallel genomic sequencing, trisomy 21, one of most common genetic
disorders featured in three 21 chromosome, is successfully detected [28]. In
this method, sequences that only match just one location in human genome are
counted and classified to each chromosome. Individual chromosome has its
own counting ratio. Due to the excessive chromosome 21, the trisomy 21 is
diagnosed by a high dose of 21 unique counting ratio compared with the
corresponding euploid reference sample. Similar methods have extended to
other aneuploidy defect detections such as trisomy 13 and 18 [29].

5. CONSTRUCT FETAL GENOME USING CELL FREE DNA


Although fetal aneuploidy could be detected only with maternal
circulating DNA using massively parallel sequencing [28], other Mendelian
diseases that severely affect the fetus remain unsolved. It seems impossible to
cover hundreds of single-gene disorders merely with a low-precision strategy.
Comparably, next-generation sequencing could provide higher throughput and
sensitivity, offering the opportunity to reconstruct fetal genome noninvasively
(table 1). Bell et. al. [30] have attempted to apply this tool in preconception
carrier screening and they scanned for 448 severe recessive childhood diseases
with just one detection.
The rationale that the second sequencing aiding noninvasive prenatal
diagnosis is based on these premises is as follows: (1) blocks of DNA, which
are long stretches or large fragments of genetic substance, are transmitted from
parents to their offspring; (2) SNP, which is a single DNA base pair position

Non-Invasive Prenatal Diagnosis ...

45

with two different alleles of the mother's genome, could be utilized to


determine the mother's two haplotypes; and (3) sequencing reads should be
numerous to permit quantitative analysis at specific genomic regions.
Table1. Prenatal detection using circulating fetal nucleic acids in maternal
plasma
Approach

Target

Potential risk

qPCR
SRY gene, RHD gene, -globin gene mutation

congenital adrenal hyperplasia [15, 16, 35],


rhesus D incompatibility [36]
-thalassemia [20,21], myotonic dystrophy[19]

SRY mRNA, CRH mRNA

Preeclampsia [23, 24]

PCR + MS
-thalassemia [26]

Disease specific DNA sequence (deletion or mutation)


SNP of PLAC4 mRNA

trisomy 21 [27]

MPGS
36 bp of each plasma DNA molecule

trisomy 21 [28, 37, 38], trisomy 18, and trisomy 13 [29]

NGS
160 average target coverage, 93% of nucleotides >20 coverage
50coverage of haploid genome, >100coverage of exome
30~coverage of genomic DNA

448 severe recessive childhood diseases [30]


whole genomic analysis [31]

whole genomic analysis [32]

Fan et al. [31] have shown a good example utilizing maternal blood
sample to reconstruct the whole fetal genome. In their study, a sample of
peripheral blood was obtained and separated into two parts: the cell part and
the cell-free part. The former was to extract the mother's DNA to directly
determine maternal haplotypes. In this step, an SNP-linked DNA block is
introduced to discriminate alleles in the mother's two haplotypes. Obviously,
longer reads of sequencing permit longer segments, mapping to the genome
and consequently more reliable results. The part of cell-free plasma contains
circulating DNA whose features we have mentioned above. When sequencing
this kind of DNA, each allele displays four portions in which two are from the
mother and two are from the fetus. As one haplotype of the fetus is inherited
from the father and the other from the mother, indeed three types of haplotype
exist in the cell-free DNA mixture (Figure 1). Because we could know
maternal whole genome sequences (two haplotypes) depending on the

46

Liang Xu and Rui Shi

sequencing of the cell part DNA extracted from the mother's peripheral blood,
when the percentage of fetal DNA is clear (usually determined by male
specific allele), we can easily deduce fetal SNP linkage alleles. Stringing these
SNP blocks together manifests the construction of the fetal chromosome
(Figure 2).

Figure 1. Fetal genome in maternal circulating DNA. A human somatic cell contains
two complete haploid sets. During meiosis, the two chromosomes of the mother or
father are recombined and randomly assorted to the offspring. As a result, three
haplotypes occur in maternal plasma: haplotype that the fetus inherited from the
mother (red color), haplotype that the fetus inherited from the father (green color), and
the maternal haplotype that is not transmitted to the fetus (yellow color).

Of note, deeper sequencing should be performed to provide enough data to


count the SNPs at individual alleles. Testing the father's DNA, even though
this is not mandatory, would be an additional reference in determining the

Non-Invasive Prenatal Diagnosis ...

47

paternal haplotype inherited by the fetus [32]. One should keep in mind that
this road map, in spite of the accurate forecast of fetal genetic secrets, is
constructed based on three premises. As a matter of fact, the average de novo
mutation rate is 1.20 x 10-8 per nucleotide per generation [33], and the fathers
age contributes to the diversity in the mutation rate of SNPs; thus the fathers
genetic information should be added to enhance the analysis of the fetal
genome.

Figure 2. Deducing fetal genome with maternal peripheral blood. Full-length


haplotypes of the mother's genome are obtained from maternal peripheral bloods, and
determined by SNP analysis. Fetal haplotypes are deduced from maternal plasma
DNA, which is a mixture of maternal and fetal DNA. During the course of fetal
genome analysis, deep sequencing of maternal circulating DNA is required to count
the specific fetal alleles.

CONCLUSION
Noninvasive prenatal diagnosis has long been considered as an ideal
examination method not only for the pregnant women but also her family. The

48

Liang Xu and Rui Shi

new-generation sequencing technology opens up a possibility of screening


hundreds of disease-susceptible genes once at the cost of only several
milliliters of blood. No one can deny that it is a great progress technically, but
it also means more caution and responsibility [34]. The more comprehensive
the genetic information about the fetus, the stricter the standard of informing
the patient right, and we should make every effort to avoid any abuse related
to decrypting the "ATCG" of the little creature we are expecting to welcome.

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[37] Ehrich M, Deciu C, Zwiefelhofer T, et al: Noninvasive detection of fetal


trisomy 21 by sequencing of DNA in maternal blood: a study in a
clinical setting. Am. J. Obstet. Gynecol. 2011; 204: 205.e1-11.
[38] Chiu RW, Akolekar R, Zheng YW, et al: Non-invasive prenatal
assessment of trisomy 21 by multiplexed maternal plasma DNA
sequencing: large scale validity study. BMJ 2011; 342: c7401.

In: Prenatal and Maternal Diagnosis


ISBN: 978-1-63482-794-2
Editor: Aaron Henderson
2015 Nova Science Publishers, Inc.

Chapter 5

ARRAY CGH IN PRENATAL DIAGNOSIS


Paola Evangelidou1, Philippos C. Patsalis, Professor 2,3
and Carolina Sismani1
1

Department of Cytogenetics and Genomics,


The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
2
Translational Genetics Team, The Cyprus Institute of Neurology
and Genetics, Nicosia, Cyprus
3
The Ministry of Health, Nicosia, Cyprus

ABSTRACT
Chromosomal abnormalities are the cause of many human genetic
disorders. Karyotyping has been for decades the golden standard method
for prenatal diagnosis and for the diagnosis of numerical and large
structural abnormalities (<3-10Mb). These, if present may result in
congenital anomalies, dysmorphism, intellectual disability, autism or
other neurological abnormalities. With the introduction of array
Comparative Genomic Hybridization (CGH) analysis in postnatal
analysis and its use as a first-tier test in cases of Intellectual disabilities, it
has been postulated that it might also become the first-tier test in prenatal
diagnosis as well.
rray CGH, a high throughput, comprehensive and fast technology,
has proven its ability to detect subtle copy number changes that can go
undetected by light microscopy. Array CGH is a valuable tool for the
determination of copy number changes in children with congenital
abnormalities and has even replaced karyotyping for certain reasons for

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani


referral. Array CGH provides valuable information for phenotypegenotype correlation, as well as more accurate information regarding the
clinical significance and the risk in the current and future pregnancy of
the respective patient. Another critical factor for accurate Copy Number
Variation (CNV) classification is parental testing to determine between
familial and de novo CNVs. Appropriate pre and post- test genetic
counceling offer the prospective parents tools to decide on the
management of their pregnancy. However, one of the problems posing
dilemmas to genetic councelors is the fact that it can detect coincidental
findings, variants of unknown significance as well as variants with
variable expressivity.
Array CGH could potentially be used in POC/intrauterine
death/stillbirths samples where malformations exist in the fetuses, using
the same platform as in prenatal cases, as it offers an increase in detection
rate in this category of samples. Furthermore, this method has the
advantage to circumvent technical problems associated with tissue
culturing which may result in failure of providing results by classical
cytogenetics.
In prenatal diagnosis chromosomal analysis is still the primary
choice of testing; for array CGH to replace it, it has to overcome some of
its limitations, such as the detection of CNVs, coincidental or of unclear
significance findings. The detection rate in clinically significant copy
number changes increases with the application of array CGH in cases
where the karyotype is normal and there are sonographic malformations
in the fetus. It therefore may be used more extensively in prenatal
diagnosis as well.
Currently there is a lot of debate whether array CGH should also be
introduced in the routine prenatal setting. However, there are still many
technical challenges to be overcome such as:

the resolution
the type of platform to be used
ethical issues including coincidental and of unclear significance
findings

A shared database specifically dedicated to prenatal diagnosis


coupled with the growing amount of data regarding CNVs and dosage
sensitive genes could make it easier to interpret genomic arrays.
Currently international consensus guidelines for the use of array CGH in
prenatal diagnosis are still missing.

Array CGH in Prenatal Diagnosis

55

1. INTRODUCTION
Prenatal Diagnosis is the application of various techniques in high risk
pregnancies to determine whether the unborn fetus or embryo is affected with
a genetic disorder or condition before birth. These genetic disorders include
syndromes caused by chromosomal abnormalities, monogenic disorders such
as cystic fibrosis, thalassemia and many others. The detection for each of these
conditions depends on the method used for diagnosis.
Chromosomal abnormalities and especially aneuploidies constitute one of
the major causes of intrauterine death [1,2] and childhood intellectual
disability [3]. Consequently, the most frequent indication for invasive prenatal
diagnosis is the detection of genetic disorders caused by chromosomal
abnormalities. As invasive procedures carry a risk for miscarriage, several
non-invasive screening tests (biochemical, ultrasound) are offered to all
pregnant women. These tests evaluate the risk of each pregnancy and only
those pregnancies classified as high risk undergo invasive prenatal diagnosis.
Invasive procedures include sampling from the placenta, the amniotic cavity
and the umbilical cord to acquire Chorionic villus (CVS), amniotic fluid (AF)
and fetal blood (FB) samples respectively.
In the last decades, chromosomal abnormalities have been detected
through conventional chromosomal analysis (karyotype), a microscopic
method that allows the identification of structural and numerical abnormalities.
Even though chromosomal analysis offers whole genome detection, this is at a
very low resolution with its detection level falling between 3-10Mb. Over the
years several techniques have emerged that complement chromosomal analysis
in prenatal diagnosis such as Fluorescence In Situ Hybridization (FISH),
Quantitative Fluorescence Polymerase Chain Reaction (QF PCR) and
Multiplex Ligation-dependent Probe Amplification (MLPA). These techniques
however are targeted and do not offer whole genome analysis as is the case
with chromosomal analysis. In recent years the development and introduction
of array CGH in clinical practice has offered an additional whole genome
approach for the detection of copy number changes. Array CGH is a high
throughput method which can be applied and detect genome wide copy
number changes at a much higher resolution than chromosomal analysis, even
as low as 1Kb. However, array CGH cannot detect balanced chromosomal
rearrangements which may also lead to a clinical phenotype. It has replaced
chromosomal analysis in postnatal diagnosis in many laboratories and is
currently used as a First-Tier clinical diagnostic test for individuals with

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

developmental delay. Its introduction to prenatal diagnosis has now increased


but it has not yet fully replaced chromosomal analysis.

2. NON-INVASIVE PRENATAL SCREENING (ULTRASOUND


MONITORING AND BIOCHEMICAL TESTING)
There are several non-invasive screening tests that can be offered to all
pregnant women. Non-invasive screens can only assess the risk of a condition
and cannot determine 100% if the fetus has a condition. Findings from the
non-invasive screening tests will determine whether or not there is a need for
an invasive prenatal procedure which will provide a diagnosis.
The non-invasive techniques include:
a) Procedures that allow fetal visualization and can be used to follow
fetal growth and detect structural abnormalities like Ultrasound, Fetal
echocardiography, MRI, Radiography
b) Listening to the fetal heartbeat
c) Screening for neural tube defects
d) Sequential screening [4,5]
In the first trimester maternal serum screening can check levels of free hCG and PAPP-A in the prospective mother's serum. These are then combined
with the measurement of nuchal translucency (NT) as well as the absence or
presence of the fetal nasal bone on the ultrasound.

3. INDICATIONS FOR PRENATAL DIAGNOSIS


As invasive prenatal procedures are associated with a risk for causing
miscarriage (estimated to be around 0.5-1 %) [5,6] it is necessary to evaluate
the absolute need for testing. Therefore it is suggested that invasive procedures
are reserved for pregnancies that are considered to be of high risk [5]. The
main indications for prenatal diagnosis are:
a) Advanced maternal age (above 35 years old). Maternal age alone is
though a poor predictor

Array CGH in Prenatal Diagnosis

57

b) Abnormal maternal serum biochemistry [for PAPP(A) and - HCG]


and/or abnormal ultrasound findings
c) Fetal anomaly detected by ultrasonography
d) Pregnancy history: previous abortus, stillbirth or livebirth with a
chromosomal abnormality (mostly aneuploidy)
e) Transmissible chromosomal rearrangement
f) Pregnant woman is a carrier of an X-Linked disorder (e.g., Fragile X)
g) Pregnant woman and partner carriers of a recessive genetic disorder
like thalassemia, cystic fibrosis etc.
h) Exposure to viral infections, such as rubella or cytomegalovirus.

4. BENEFITS OF PRENATAL DIAGNOSIS


Invasive prenatal testing can be carried out earlier or later in pregnancy
and it can offer the future couple the most suitable obstetric management by
having:
a) The choice to decide on the outcome of the pregnancy once the
genetic or other result is available
b) Help in determining whether to continue the pregnancy
c) An estimate of the complications in the pregnancy and birth
d) Preparation of the couple for the birth of a child with an abnormality,
and potentially offer education about the specific disorder and
preparation for the special care that will be required of a handicapped
child
e) A prognosis for future pregnancies for themselves and/or their
immediate and extended families.
Therefore clinicians and patients should weigh the relative risks and
benefits of invasive prenatal diagnosis performed later as compared to earlier
in pregnancy.

5. TYPES OF INVASIVE PROCEDURES


For decades, Chorionic Villus Sampling and Amniocentesis have been the
two most common prenatal diagnostic procedures. Both are invasive

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

procedures requiring the need of a needle being passed through the cervix or
through the abdominal wall into the uterus under ultrasound guidance. A
sample of chorionic villi surrounding the sac is obtained for CVS.
Altrernatively 10-20 mL of amniotic fluid (AF) from the amniotic cavity
inside the uterus is collected for amniocentesis.
Chorionic Villus Sampling is collected during the first trimester of
gestation (11-14 weeks) and once collected the villi are dissected under an
inverted microscope from the maternal decidua. CVS contains chorionic villi
which are microscopic, finger-like projections that emerge from the chorionic
membrane and eventually form the placenta. The cells that make up the
chorionic villi are of fetal origin. Following an enzymatic dissociation of the
sample it is set up into cultures to eventually harvest metaphase cells
(fibroblasts) for chromosomal analysis to be carried out.
Amniocentesis is carried out either early on the second trimester (15-27
weeks) or late in the third trimester (28weeks -to term). The Amniotic Fluid
(AF) contains a heterogeneous population of cells and depending on the
gestational age they are arising from the amnion, skin and the urogenital or
respiratory tract.
The numbers of fetal cells present in the AF sample increase with
gestational age, but the viable cells are decreasing in numbers as the
pregnancy progresses. Usually 10-20 ml of amniotic fluid is collected and
presented to the laboratory for chromosomal, biochemical, and/or molecular
analyses. The AF is set up into cultures to eventually harvest metaphase cells
(fibroblasts) for chromosome analysis to be carried out.
Alternatively, for both CVS and AF samples, DNA can be extracted for
molecular analyses like QF PCR or array CGH to be carried out.
Both procedures are safe with an equivalent risk of 0.5% of procedureinduced pregnancy loss7. It has been demonstrated by several studies that with
equally experienced operators, CVS and second trimester amniocentesis have
similar procedure-induced miscarriage rates [7]. As is mentioned in R.
Wapner 2005 7 when CVS procedures are performed after 10 weeks gestation,
no increased risk of fetal anomalies has been demonstrated; on the contrary,
when CVS is carried out prior to 10 weeks of gestation there may be an
increased risk for limb reduction defects; likewise when the amniocentesis is
done prior to the 15 weeks it has an increased risk for talipes equinovarus.
Laboratory analysis for both procedures is equally reliable.
When carrying out chromosomal analysis from CVS, the karyotype is
identical to that of the fetus in over 98% of cases. In the remaining 1 to 2%
confined placental mosaicism (CPM) occurs and therefore there might be a

Array CGH in Prenatal Diagnosis

59

need for a second invasive procedure to exclude confined placental


mosaicism [7].
First-trimester CVS has an advantage over second-trimester
amniocentesis, in that it allows earlier prenatal diagnosis of various genetic
and cytogenetic disorders in the fetus, thus giving the prospective couple the
choice of earlier termination should it decide that to be the outcome of the
pregnancy [8].
Test results for amniocentesis, whether done earlier or later in pregnancy,
are usually available only after the 18th week of gestation. Therefore CVS has
developed to avoid the medical and psychological complications of later
prenatal diagnosis by amniocentesis. In many countries CVS has rapidly
become a primary tool for the diagnosis of fetal cytogenetic, molecular, and
biochemical disorders. In addition, its development has led to an improved
understanding of several biological processes, including confined placental
mosaicism and uniparental disomy [9].
Fetal Blood (FB) sampling, also known as cordocentesis, is the third type
of invasive prenatal diagnosis. It is performed after the 16th week of gestation
and the sample is acquired through the insertion of a needle into the umbilical
cord under ultrasound guidance. Fetal blood is the sample that is the most fetal
in origin and it would be the most reliable material to use. Unfortunately since
the availability of sampling expertise is insufficient and the obstetric
complications are relatively high, it is not always possible for it to be obtained.
When acquired though, its advantage is the rapid rate at which lymphocytes
grow, allowing prompt genetic diagnosis. DNA can be extracted from whole
(fetal) blood for other molecular analyses. Another disadvantage of fetal blood
sampling is that it is performed after the 16th week of gestation. Results
however can be achieved much earlier compared to CVS and AF.
Based on the fact that the constitutional karyotype of an individual is
determined at conception, and mitosis just copies this genotype in all tissues
derived thereafter, by karyotyping cells from different tissues the fetal
karyotype can be determined [10]. In 99% of cases the fetal karyotype is
indeed reliably detected. The remaining 1% of the cases is more problematic
due to mosaicism. Mosaicism occurs when there is a mitotic error early in fetal
life. Depending on the stage of fetal development it takes place, mosaicism
will be apparent in the placenta, or the extra-embryonic tissues or even the
embryo.

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

6. EVACUATED PRODUCTS OF CONCEPTION (POC)


In the event of spontaneous miscarriage of a pregnancy, evacuated
products of conception could be sent to the laboratory to determine if the
miscarriage had occurred due to genetic abnormalities. When this type of
sample is received by the laboratory an effort is made to retrieve fetal material
so that cultures can be set up for chromosomal or other molecular analyses. If
this is a freshly first trimester miscarriage there are good chances of finding
intact limbs from the fetus. These could be selected for culture as they will
grow very well. Most of the times however, these samples do not contain any
obvious fetal parts, therefore the laboratory should try and isolate anything that
is truly fetal. Good substitutes in this case would be chorionic villi, embryonic
sac or membranes. A major disadvantage of POC samples is the fact that along
with the presumably fetal material, maternal tissue is also collected which can
also grow in culture. This will interfere with the interpretation of the results. In
addition, a number of these kinds of samples fail to grow in vitro or
bacterial/fungal contamination occurs. This makes it impossible to conclude
the analysis, in which case DNA is extracted from the same tissue that was
used for setting up cultures, for analysis with alternative molecular methods.

7. ABNORMALITIES DETECTED IN PRENATAL DIAGNOSIS


7.1. Numerical Abnormalities
The most frequent abnormalities in prenatal diagnosis are numerical, when
instead of the normal two copies of chromosomes there are 3 copies (trisomy)
or only one copy (monosomy). Four copies or five copies of chromosomes can
exist at times but this mostly involves the sex chromosomes. Most numerical
abnormalities are non-viable. Viable autosomal monosomies (monosomy 21
was the only one detected from our experience of over 20 years in 3 cases) are
extremely rare and certain autosomal trisomies that can be viable are very
frequently lost during pregnancy. Sex-chromosome aneuploidies are in general
the ones that are mostly more tolerated. Approximately 30% of affected
fetuses of gestational age between 12 weeks and term will miscarry. In
addition the estimated rate of lethality between 16 weeks and term is 20% [4].
It is very rare for any other autosomal trisomy other than 13, 18 and 21 to
survive through or even near to term with an exception of mosaic or partial

Array CGH in Prenatal Diagnosis

61

trisomies, for chromosomes 8, 9 and 12. Table 7.1 shows the association of
certain numerical abnormalities, encountered prenatally, with sonographic
markers and maternal serum biochemistry.
Table 7.1. Association of chromosome abnormalities with sonographic
markers and maternal serum biochemistry at the 11-14 week gestational
age scan according to Cicero et al. 2003 [5]
Abnormality

HCG
(2MoM)

PAPP- A
(0,5 MoM)

Trisomy 21

Increased

Decreased

Trisomy 18

Decreased

Decreased

Trisomy 13

Decreased

Decreased

Turner
syndrome
Triploidydiandric

Normal

Lower

Greatly
increased

Mildly
decreased

Triploidydigynic

Markedly
decreased

Markedly
decreased

Sonographic markers
Increased NT, absent nasal bone in
60-70% of trisomies
Early onset IUGR, relative
bradycardia, associated exomphalos in
30% of the cases
Fetal tachycardia in 60% of cases,
early onset IUGR, holoprocencephaly
or exomphalos in 30% of cases
Fetal tachycardia in 50% of cases,
early onset IUGR
Early onset asymmetrical IUGR,
relative bradycardia,
holoprocencephaly, exomphalos or
posterior fossa cyst in 40% of cases,
molar changes in the placenta in 30 %
of cases
Early onset asymmetrical IUGR,
relative bradycardia,
holoprocencephaly, exomphalos or
posterior fossa cyst in 40% of cases,,
molar changes in the placenta in 30 %
of cases

7.2. Structural Abnormalities


Structural aberrations are the result of chromosomal breaks that occur
during meiosis. They can affect one or two chromosomes and they can be
balanced or unbalanced.

7.2.1. Balanced Rearrangements


In balanced structural abnormalities (translocations, inversions, insertions)
there is no gain or loss of genetic material. The major consequence of a
balanced rearrangement is however the prevention of normal chromosome
pairing at meiosis, which leads to production of sperm and eggs with

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

incomplete or partially duplicated chromosome sets. Although most carriers of


balanced translocations are phenotypically normal, an association of
cytogenetically balanced translocations with phenotypic abnormalities has
been reported [11]. The main mechanisms leading to a phenotype are:
i)
ii)
iii)

iv)

v)

The disruption of a dosage-sensitive gene at the breakpoints or


expression a recessive gene
Position effect with variable expression of genes near the
translocation breakpoint
Uniparental disomy (if the chromosome involved is subjected to
imprinting) due to post-conceptional correcting loss of the homolog
from the normal non-carrier parent
The rearrangement is not truly balanced at the DNA level or in
familial cases there may be additional unbalanced subtle
rearrangements which occurred during meiosis
The rearrangement may host cryptic complex chromosomal
rearrangements (CCRs).

The clinical significance of prenatal and postnatal identification of cryptic


CCRs is extremely important, as CCRs are associated with reproductive
problems, multiple miscarriages, stillbirths or with malformations, intellectual
disability, dysmorphic features or congenital anomalies [12].

7.2.1.1. Translocations
Balanced reciprocal translocations are produced by the interchange of
parts of two chromosomes without visible loss of chromosomal material.
When the translocation involves the acrocentric chromosomes it is termed a
Robertsonian Translocation. In the normal population 1 in 500 people carry a
balanced rearrangement [13]. The great majority of apparently balanced
translocations are usually not associated with abnormal phenotypes. There is a
risk of phenotypic abnormalities however in 6.1% of de novo apparently
balanced translocation carriers [14]. In conventional cytogenetics a
translocation may seem apparently balanced, but studies have shown that this
is not always true. Even if a translocation is confirmed by FISH analysis using
subtelomeric specific and whole chromosome paints, it may not always be
truly balanced. Sismani et al. [15] demonstrated by array CGH that 3 out of 12
(25 %) postnatal balanced translocation cases, both familial and de novo, with
abnormal phenotype, carried cryptic imbalances near the translocation
breakpoints or on another chromosome unrelated to the translocation. In a

Array CGH in Prenatal Diagnosis

63

prenatal study of 25 fetuses with normal or balanced karyotype and abnormal


ultrasound findings we showed that the use of array CGH revealed copy
number changes in 3 out 25 cases (12%,) two of which (8%) were considered
clinically significant. One of these cases was an apparently balanced
translocation case and the deletion was located at the translocation breakpoint
[16].

7.2.1.2. Inversions
An inversion occurs when there are two breaks on a single chromosome
and a 180 degree rotation of the section between the breaks. The breaks could
either take place on the same arm (Paracentric inversions) or on different arms
(Pericentric Inversions). Inversions can be identified by conventional
cytogenetics and are usually confirmed by FISH.
7.2.1.3. Insertions
An insertion occurs when chromosomal material from one chromosome is
inserted (inverted or in the same direction) at a different point of the same
chromosome or on another chromosome. This is an apparently balanced
rearrangement and it can be detected by classical cytogenetics.
7.2.2. Unbalanced Rearrangements
Unbalanced rearrangements (deletions, duplications) are very similar to
numerical abnormalities, with the difference that there is only partial and not
complete gain or loss of a chromosome. This partial gain or loss could include
large or smaller parts of chromosomes. But even if the copy number gain or
loss is a tiny chromosome fragment it could have severe effects to the
phenotype as it encompasses several genes. This is clearly demonstrated in
cases where the subtelomeric regions are involved as well as in
microdeletions/microduplications syndrome regions.
7.2.2.1. Deletions
Deletions occur when there is loss of genetic material and they can be
terminal or interstitial. Subtle interstitial deletions (<3-5 Mb) are called
microdeletions and many of them have been associated with specific
syndromes (DiGeorge/ Velocardiofacial syndrome, Smith Magenis, MillerDieker etc.). If a patient is suspected of carrying one of these syndromes the
physician will refer them for targeted investigation of that particular syndrome
with locus specific FISH (for example the 22q11.2 deletion syndrome). These
deletions are difficult to be detected even with extended chromosomes and

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

might be missed by classical cytogenetics. Terminal deletions when suspected


at classical cytogenetics analysis could be confirmed by subtelomeric FISH
analysis. Otherwise small terminal deletions that escape chromosomal analysis
will remain unidentified.
In a large study, by Knight et al., it was demonstrated that rearrangements
in the subtelomeric regions are associated with intellectual disability, as they
occurred in 7.4% of the moderately to severely affected individuals and 0.5%
of the mildly affected [17].
Finally interstitial deletions that exist on locations other than those of the
microdeletion syndromes, if really small (<3-5 Mb) could again remain
undetected by cytogenetic analysis. This is the main advantage of array CGH;
a single assay that can detect all possible abnormalities present in a patient that
cannot be identified by classical cytogenetics. Follow up is of course necessary
with such findings as they need to be confirmed in the patient and the parents
to determine their clinical significance and whether they are de novo or
familial, so that the recurrence risk can be estimated.
Bateman et al. and Filges et al. report on cytogenetically visible interstitial
deletions one de novo and one familial respectively of no phenotypic effect
[18,19]. In the de novo case the patient had a 9.3Mb-10.7Mb deletion, was of
normal intelligence, had no dysmorphic features and had experienced 3
miscarriages. In the familial case there was a 14.5Mb deletion in three
generations with no relevant phenotypic effect. Follow up is important and can
usually be carried out with the application of array CGH in the parents and/or
FISH analysis in the patient and the parents.

7.2.2.2. Duplications
Duplications occur when a section of genetic material is duplicated.
Duplications can be seen cytogenetically and can be identified as the
duplicated material appears in tandem, in the same or in an inverted
orientation. The exact location of duplications identified by array CGH cannot
be known. The use of FISH would be needed in order to determine the
physical location of the duplicated segment [20]. This is very important in
diagnosis especially for de novo duplications in affected children where the
duplication could mean the presence of an insertional translocation in one of
the parents [21]. The clinical significance of this finding lays with the
determination of the recurrence risk in future pregnancies.

Array CGH in Prenatal Diagnosis

65

7.3. Uniparental Disomy (UPD)


Uniparental Disomy (UPD) is the presence of a chromosome pair derived
from one parent in a disomic cell line [22]. UPD is considered an important
diagnostic [23] and prognostic factor for special syndromes [24,25]. There are
three major types of UPD:
1) for the entire chromosomal complement
2) for a complete chromosome
3) segmental UPD.
UPD is also subdivided into:
1) heterodisomy (hUPD); the inheritance of both chromosomes from one
parent
2) isodisomy (iUPD); the inheritance of two copies of the same
chromosome from one parent.
UPD of the entire chromosomal complement, either maternal or paternal
in origin, leads to benign cystic ovary and complete hydatidiform mole
respectively [26]. UPD for a complete chromosome or a segment of a
chromosome may cause a disease if they are affecting a gene underlying
genomic imprinting. Examples of Syndromes caused by UPD include Prader
Willi syndrome [MatUPD(15), OMIM #176270], Angelman Syndrome
[PatUPD(15), OMIM #105830] Silver- Russel syndrome [ MatUPD(7) OMIM
# 180860] and others. In addition, iUPD can cause a recessive disease in the
offspring of a carrier parent [22].
Twenty years ago it was thought that UPD would be a rare event, but
today there are more than 1,100 UPD clinical cases in the literature [27]. The
frequency of UPD in newborns is considered to be about 1 in 3,500 births- a
rate of 0.029% [28]. In a large number of cases UPD is identified in
connection with a chromosomal abnormality such as a Robertsonian
translocation an isochromosome or a small Supernumerary Marker
chromosome. Segmental UPD arises due to postzygotic somatic recombination
between maternal and paternal homologues or with numerical and/or structural
abnormalities (partial trisomies for example) [27]. UPD can only be detected
with MLPA, Microsatellite analysis and SNP arrays.

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8. CURRENT METHODOLOGIES IN INVASIVE


PRENATAL DIAGNOSIS
8.1. Cytogenetics-Classical Chromosomal analysis
Cytogenetics includes routine analysis of G banded chromosomes, and/or
other cytogenetic banding techniques (C- Banding, NOR, Q Banding), as well
as molecular cytogenetics, such as Fluorescence In Situ Hybridization (FISH)
and metaphase Comparative Genomic Hybridization (mCGH). The aim of
Classical Cytogenetics is to count and to structurally analyze the chromosomes
for phenotype- genotype correlation, or for prenatal referrals to correlate
sonographic markers or other indications to the genotype (Figure 8.1).

Figure 8.1. G- Banded male karyotype from an amniotic fluid sample showing 46
chromosomes.

Cytogenetics has been used since 1970 for prenatal diagnosis and is still
used as the primary detection method for prenatal samples. Chromosomal
analysis is usually carried out at the 550 Band Level (G-Banding) and offers
whole genome detection at a very low resolution with its detection level falling

Array CGH in Prenatal Diagnosis

67

between 3-10Mb. It can successfully identify structural abnormalities either


balanced or unbalanced. Rearrangements smaller than 3Mb, or even larger
rearrangements that occur in regions where the banding pattern is not
distinctive (i.e the subtelomeric regions), are undetectable with this method.
They could however account for a substantial fraction of the intellectual
disability referrals.
Some chromosome abnormalities maybe harmless variations [29], but
most are associated with clinical disorders [30,31]. Half of all first trimester
spontaneous abortions are due to chromosome abnormalities but the incidence
in live births falls to less than 1 per cent. Loss or gain of whole chromosomes
can cause severe disorders as it can affect the copy number of thousands of
genes.
Chromosomal analysis has an increased turnaround time, it is time
consuming and requires highly skilled staff. In addition it may not identify the
origin of supernumerary marker chromosomes or ring chromosomes present in
the analysis. Use of other methods is required to further investigate them.

8.2. Molecular Cytogenetics- Fluorescence In Situ Hybridization


(FISH)
FISH is a targeted, molecular cytogenetics technique that uses
fluorescently labeled DNA probes to hybridize to the specific locus of interest.
These include subtelomeric specific, whole chromosome paint, locus specific
or centromeric probes. FISH can be used as a diagnostic method for the
microdeletions/microduplications syndromes and the subtelomeric regions that
are beyond the resolution of conventional cytogenetics. It can also be used as a
rapid aneuploidy testing in interphase nuclei in Prenatal Diagnosis,and serve
as a complimentary method to conventional cytogenetics or array CGH
analyses.
Currently in prenatal diagnosis the only microdeletion which has specific
symptoms and recognizable ultrasound findings during fetal life (conotruncal
cardiac defect) is the 22q11.2 causing DiGeorge/ Velocardiofascial syndrome
(VCFS) [32]. There is a large number of microdeletion and microduplication
syndromes known today. Most of these however remain undetected until after
birth due to the fact that they have non- specific or no symptoms at all during
fetal life [33]. Array CGH could be performed prenatally and detect these
conditions prior to birth.

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

8.3. Quantitative Fluorescence Polymerase Chain Reaction (QF


PCR)
QF PCR was first reported in the early 1990s. It is a rapid aneuploidy
detection method for the common aneuploidies (chromosomes 13, 18, 21, X
and Y) and is usually carried out in conjunction with chromosomal analysis. In
QF PCR highly polymorphic Short Tandem Repeats (STRs) for these
chromosomes are amplified using fluorescence primers and PCR in a
multiplex assay, followed by automated analysis of the fluorescence intensity
of the alleles in a genetic analyzer [34]. These aneuploidies account for more
than 80% of clinically significant chromosomal abnormalities diagnosed in the
prenatal period [35]. QF PCR can identify the presence of maternal
contamination [7] in the processed samples which could serve as a tool later
on in the samples used for array CGH. QF PCR cannot detect mosaicism of
less than 20-30% and it is designed to identify only the abnormalities that are
specifically looked for. There is a residual risk of chromosome aberration after
QF PCR (or FISH) show a normal result. This risk was estimated to be 0.9%
for all indications for invasive prenatal diagnosis and in 0.4% of all the
invasive tests the chromosome aberration was of clinical significance [36].

8.4. Multiplex Ligation-Dependent Probe Amplification (MLPA)


MLPA methodology was initially described in 2002 by Schouten et al.
[37]. It is used to detect abnormal copy number changes based on relative
quantification of probes specifically designed for the regions of interest. It
allows simultaneous detection of abnormal copy numbers for up to 40-50
regions. MLPA is based on multiplex PCR methodology using fluorescently
labeled universal primers and relies on probe amplification of different size
probes rather than amplification of the genomic test DNA. MLPA is
performed in solution and the probes are designed in 2 parts and are dependent
on the presence of the targeted region for ligation and creation of a contiguous
probe to be amplified. Hybridization/Amplification ratios are compared with
control regions [37].
MLPA is used in prenatal diagnosis as an alternative method to QF-PCR
for the detection of aneuploidies of chromosomes 13, 18, 21, X and Y. In
addition, it is also widely used for the detection of copy number changes in
regions of interest such as the microdeletion/microduplication syndromes
region and many others [38].

Array CGH in Prenatal Diagnosis

69

8.5. Metaphase CGH (mCGH)


Before array CGH was implemented into clinical practice metaphase
Comparative genome hybridization mCGH was widely used since 1992 [39],
especially in cancer cytogenetics. It was based on in situ hybridization of
differentially labeled patient DNA and normal reference DNA to normal
human chromosome spreads. Briefly, DNA is extracted from a control
individual with a known, normal karyotype and a testing individual with an
unknown karyotype. These two DNA samples are differentially labeled with
two different fluorochromes and applied to metaphase spreads of a normal
human. After hybridization the intensity ratio between the patient and normal
fluorescence is measured and copy number variations in the patient DNA are
detected.

9. A NEW ERA IN CYTOGENETICS AND PRENATAL


DIAGNOSIS WITH THE USE OF ARRAY CGH
This revolutionary technology was first developed as a research tool for
the investigation of genomic alterations in cancer [40]. Array CGH compares
DNA content from two differentially labeled genomes, a test/patient and a
reference/control. After labeling, these two genomes are co-hybridized onto a
solid support, usually a glass microscope slide, on which cloned or synthesized
DNA fragments are immobilized.
Arrays have been developed in a variety of designs over the years. They
have been constructed to span the whole genome and they use various-sized
targets from Bacterial Artificial Chromosome (BAC) to synthetically
synthesized oligonucleotides. Each BAC or oligonucleotide has a known
position within the human genome.
One of the first arrays used, was the 1 Mb BAC array with 1Mb backbone
with additional BACs at regions known to be involved in the major human
genetic disorders. Whole genome array also included the Tiling path BAC
array consisting of 26,574 clones and covering 93.7% of euchromatic regions
introduced by Fiegler et al. [41]. In addition to the whole genome arrays,
targeted arrays also exist for specific regions of the genome. It could be
targeted to study a specific chromosome [42], or chromosome segment [40] or
to detect and identify specific DNA dosage abnormalities in individuals with
suspected microdeletion syndromes [43] or subtelomeric rearrangements [44].

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

The resolution of the array is defined by the size of the nucleic acid target on
the array and the density of the coverage over the genome. The smaller the
nucleic acid sequence and the more contiguous/overlapping the targets are, the
higher the resolution of the array will be.
The arrays have been designed to provide redundancy with high
sensitivity and specificity for the detection of clinically significant genomic
imbalances.
In addition to BAC and oligonucleotide arrays, Single Nucleotide
Polymorphism (SNP) arrays are also available by several manufacturers.
The resolution of array CGH has the potential of being much higher than
that of conventional cytogenetics as it is determined by the size of the target
arrayed on the solid platform and the coverage or density of those targets.
Array CGH has the ability, as mentioned previously, to investigate
simultaneously thousands, or even more, loci on a single assay. This is an
advantage of array CGH over classical cytogenetics and FISH. However, as
Shaffer and Bejjani very well discuss, microarray analysis is not a standalone test in the diagnostic laboratory as other methodologies are needed to be
carried out in order to be able to determine the chromosome rearrangement
that occurred in the discovery of a copy number change [45]. This is
extremely important, in the discovery of an aberration in a child, in order to be
able to offer parents counseling for the condition of their child as well as for
recurrence risk.
In addition, array CGH analysis has revealed that many familial DNA
gains or losses across the genome are abundant [46]. Some of these Copy
Number Variants (CNVs) are of no clinical significance as they have been
seen in both phenotypically normal and abnormal individuals. Others are
believed to have clinical significance. And finally a number of CNVs cannot
be classified as more cases with the same genomic imbalances need to be
identified and evaluated in order to categorize them as being causative or not.
The discovery of CNVs in a diagnostic setting creates problems to the
analyzers as they need to go through publicly available or in house databases
in order to determine whether the CNVs found have any clinical significance.
Another limitation of array CGH is the potential number of CNVs that
will require further investigation prior to the final result. Further investigation
would mean further testing of the patient and the parents with array CGH,
FISH or other molecular methods. This in turn will mean longer turnaround
times for the final result to reach the patient and higher costs. In prenatal
diagnosis longer turnaround times will mean longer periods of anxiety for the
prospective parents.

Array CGH in Prenatal Diagnosis

71

9.1. Microarray Comparative Genomic Hybridization


(Array CGH)
In contrast to mCGH, array CGH permits a more detailed analysis with
refined resolution even at the level of genes. The fundamental principle is the
same as mCGH. It is a comparative genomic hybridization using array rather
than a metaphase spread as a substrate. Array CGH requires for the test and
reference DNA of the same gender to be co-hybridized on the array of choice,
as previously described [41]. Briefly, patient and reference DNA are
differentially labeled and are then hybridized on arrays. Array images are then
acquired using a scanner and image files are quantified and analyzed using
appropriate software [41].
The microarray is comprised of thousands of spots of reference DNA
sequences, applied in a precisely gridded manner on a slide. The resolution of
the array CGH depends on how many spots of reference DNA exist on the
slide. A slide with 3,000 spots would have a resolution of 1 Mb across the
entire genome. These spots could be Bacterial Artificial Chromosome (BAC)
clones, Oligonucleotides, cDNA. Nowadays the resolution of array CGH has
significantly increased as new microarrays might include up to two million
oligonucleotides. Array CGH can detect gains and losses of very short
genomic segments at multiple loci in a genome in a single assay, which gives
it an advantage over conventional cytogenetics and FISH. Array CGH has
some limitations however as it cannot reveal any balanced rearrangements,
polyploidy, mosaicism below 20% and small or point-recessive mutations.

9.2. Array CGH in Prenatal Diagnosis


Array CGH is increasingly performed for the evaluation of individuals
with birth defects, dysmorphic features and mental retardation. Genome-wide
arrays are rapidly replacing conventional karyotyping in postnatal diagnostics
and some studies suggest that it should be used as a First-Tier clinical
diagnostic test for individuals with developmental delay [47]. Array-CGH is
currently also used in prenatal diagnosis quite extensively. This however can
only be done in conjunction with chromosomal analysis.
When applying array CGH in prenatal diagnosis, certain issues should be
addressed:

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

for which pregnancies array CGH should be carried out


whether it will be for all pregnancies or for pregnancies with
ultrasound abnormalities
which array platform to use
to define the appropriate calling criteria
which methods will be used to confirm array CGH findings and pretest counselling.

Pre-test counseling is especially important in the prenatal setting. It should


be carried out to inform parents of the possibility of the fortuitous discovery of
a CNV unrelated to the phenotype during array CGH analysis. It should be
explained to the parents that there may be asymptomatic/pre-symptomatic
results with array CGH analysis and they should be free to decide whether
they wish to be informed of these findings or not [48].
The major challenges faced in the routine diagnostic clinical setting after
the implementation of the array CGH methodology involve primarily:

the interpretation of the results


confirmations
incidental findings
polymorphisms
incomplete penetrance and variable expression of certain copy
number changes
intra-familial variability
mosaicism
availability of parental samples
genetic counseling

Copy number variants (CNVs) are unexpectedly common in the human


genome and many are without apparent clinical consequence [49]. Classifying
a copy number change as pathogenic or benign is not always straight forward.
Careful assessment of the available databases of pathogenic and benign CNVs
(e.g., DGV, DECIPHER, UCSC), and analysis of parental samples to
determine whether the CNV is de novo or inherited are required. Phenotypic
variability and incomplete penetrance of certain copy number changes
inherited from apparently unaffected parents must always be considered when
interpreting array data. Very often a CNV is of unclear clinical significance
and classification is not possible. This is one of the reasons that the use of

Array CGH in Prenatal Diagnosis

73

array CGH in prenatal diagnosis is limited. Even though array CGH has
distinct advantages over conventional cytogenetics, as mentioned previously,
this technology cannot, in our opinion, currently replace classic cytogenetics in
prenatal diagnosis. There are some laboratories however that have moved to
array CGH analysis as a first line test in prenatal diagnosis either in parallel
with conventional cytogenetics [50] or in conjunction with QF PCR [51].
Srebniak et al. suggest that array CGH is offered for all invasive prenatal
testing analysis using the same array platform, but using different resolution in
analysis depending on the reason for referral [52]. They suggest a high
resolution analysis to be used for cases with ultrasound findings but a lower
resolution of 0.5Mb for all other indications [52].
One challenge for the future will be to perform non-invasive array CGH
on free fetal DNA isolated from maternal circulation. It was previously
demonstrated that array CGH can be performed on very small amounts of
DNA with or without whole genome amplification [53]. In this way it may
enhance the potential for success of prenatal diagnosis from noninvasively
sampled fetal DNA, either as cell-free DNA from maternal plasma or blood
[54], or as fetal cells from the cervix [55].

9.2.1. Array CGH Detection Rate in Prenatal Diagnosis


Many groups have demonstrated that by applying array CGH in prenatal
diagnosis there was an additional detection of clinically significant genomic
imbalances of 3.6% when the karyotype was normal. This was regardless of
the indication of the referral for chromosomal analysis. This detection rate
increased to 5.2% when the pregnancy had a structural malformation on
ultrasound [56-63]. In these studies the overall detection of array CGH over
chromosomal analysis was 12%. When benign CNVs were removed and
considered as normal results the detection rate dropped to 3.6% [64]. This
percentage included the pathogenic CNVs as well as the Variants of Unknown
Significance (VOUS) with the potential of being pathogenic. The presence of
VOUS was found in 1.1 % of cases [64].
As mentioned above the detection rate increased for the cases where the
referral included ultrasound abnormalities and a normal karyotype. In these
studies the overall detection of array CGH was 11.2%. When benign variants
were excluded and included in the normal results the detection rate dropped to
5.2% [57, 59-63]. Furthermore, the presence of VOUS was in 1.9% of the
studies. The ultrasound findings included cardiac abnormalities, increased
nuchal translucency, cystic hygroma or hydrops or central nervous system
abnormalities. Most of these studies used Targeted BAC arrays [56-61, 63]

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

and some used both targeted and whole genome arrays [57, 58, 63]. The
resolution for the arrays varied from 287 to 4685 BAC probes and 44,000 to
946,000 oligonucleotide probes.
Tyreman et al. conducted a retrospective analysis of 106 karyotypically
normal referrals with ultrasound findings using the GeneChip 6.0 SNP array
from Affymetrix. This platform provides uniquely high resolution coverage of
the genome with over 1.8 million probes, using oligonucleotide targets that
provide copy number information only and Single Nucleotide Polymorphisms
(SNPs) oligonucleotide targets which provide genotyping as well as copy
number information. In this study a total of 35 rare CNVs were identified, 10
(9%) of which were considered to be pathogenic, 12 were likely to be benign
(11%) and 13 were VOUS (12%). The percentage of VOUS is slightly higher
than the other studies because parental testing was not used in this study. In
addition, in this study a case with a cryptic mosaic trisomy for chromosome 10
was identified as well as a case with Loss of Heterozygosity (LOH).The same
platform can detect triploidy as well which is a major advantage. One of the
limitations of array CGH is its inability to detect triploidies [62]. Table 9.2.1
shows the comparison between these studies.
In another study completed by Fiorentino et al. [65] pregnant women were
referred for chromosomal and array CGH analyses. Both methods were carried
out concurrently in order to compare results. A total of 1,037 prenatal samples
were studied and the reason for referral of these samples included advanced
maternal age, ultrasound findings, parental anxiety and family history of a
genetic condition or chromosome abnormality. Array CGH was carried out
using whole-genome BAC array with a resolution of 1Mb across the genome
and ~100kb resolution in 139 regions associated with constitutional disorders.
From the analysis it was determined that 13% of the samples had likely benign
and of no clinical significance CNVs.
Furthermore, array CGH revealed clinically significant chromosome
alterations in 3.3% of the samples. In 0.9% of the samples, array CGH
provided diagnosis of clinically significant chromosomal abnormality which
was not detected by chromosomal analysis and would have otherwise gone
undetected. Clinically significant results were also identified by conventional
cytogenetics as well as in 73.5% of the total abnormalities also detected by
array CGH (25/34) and in 2.4% of the total number of samples. In a
subsequent study Fiorentino et al. demonstrate the utility of array CGH as a
fist-line diagnostic test for all pregnant women who underwent invasive
prenatal testing, irrespective of the reason for referral [50].

75

Array CGH in Prenatal Diagnosis

The use of array CGH showed an increase of detection rate compared to


traditional karyotyping, regardless of the indication for analysis.
Table 9.2.1. Comparison between various studies which used array CGH
in Prenatal diagnosis

Study

Array Type

Kleeman
et al., 2009
[63]

Signature
prenatal targeted
BAC chip V,
signature whole
genome chip
Targeted
Genosensor
BAC/PAC array

Vialard
et al., 2009
[59]

Bi et al., 2008
[57]

BCM V6
oligonucleotide
array

Shaffer
et al., 2008
[60]

Prenatal targeted
BAC array

Sahoo et al.,
2006 [56]

BCM V4 targeted
BAC array

Tyreman
et al., 2009
[62]

GeneChip SNP
whole genome
oligonucleotide
array
Signature V 4.0,
prenatal targeted
BAC array and
whole genome
array

Coppinger
et al., 2009
[58]

Karyotype/
Reason for
Referral
Normal
karyotype,
sonographic
anomalies

Results

Clinical Significance
of Results

4/50 abnormal

2% clinically
significant, 6%
inherited or benign
variant

Normal
karyotype,
multiple
congenital
abnormalities
Normal
karyotype,
maternal age,
sonographic
anomalies,
family history,
miscarriages
149/151 normal
karyotype,
maternal age,
sonographic
anomalies,
family history,
parental anxiety
93/98 normal
karyotype,
maternal age,
sonographic
anomalies,
family history
sonographic
abnormalities

4/37 abnormal

10.8% clinically
significant

3/15 abnormal

13% clinically
significant, 7%
inherited or benign
variant

15/151
abnormal

1.3% clinically
significant, 8%
benign, 0.5% unclear
significance

5/98 abnormal
of which one
had additional
abnormalities

5% clinically
significant

35/106
abnormal

Normal
karyotype,
maternal age,
sonographic
anomalies,
family history,
anxiety

Whole
genome:
22/180
abnormal.
Targeted: 7/62
abnormal

9% likely pathogenic,
12% likely benign,
13% unclear
significance
Whole genome: 2.7%
clinically significant,
0.5% unclear
significance, 8.8%
benign variants.
Targeted: 0.9%
clinically significant,
0.5 unclear
significance, 8%
benign variants

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani


Table 9.2.1. (Continued)

Study

Array Type

Fiorentino
et al., 2011
[65]

Cytochip Focus
BAC array

Scott et al.
2013 [51]

Targeted version
of 8x60K
oligonucleotide
Agilent array

Evangelidou
et al.,
2013[66]

Oligonucleotide
arrays 105K or
180K

Karyotype/
Reason for
Referral
Maternal age,
sonographic
anomalies,
family history,
anxiety
Maternal age,
sonographic
anomalies,
family history,
anxiety

60/64 Normal
and sonographic
anomalies,
Balanced
karyotype with
or without
sonographic
anomalies,
4/64 Abnormal
karyotype or
MLPA

Results

Clinical Significance
of Results

34/1037
abnormal

3.3% clinically
significant, 13%
benign variants.

156/1049
abnormal

11.7%
Numerical/Structural
abnormalities,
3.1% CNVs
undetectable by GBanding of which:
1.2% pathogenic,
0.2% VOUS,0.9%
benign, 0.57% carrier
status of disease
11% Clinically
significant changes
15.6% VOUS
determined to be
familial CNVs
unrelated to the RFR
in 100%

17/64
Abnormal
4/17
Confirmation
of previous
abnormal
results

Furthermore, in the same study [50] it was recommended that in order to


limit the number of VOUS detected, a more careful selection of platform
should be opted with higher coverage in the known constitutional disorders
location and less coverage in polymorphic CNVs and in backbone (compared
with oligonucleotide or SNP arrays).
Scott et al. first reported on the effective combination of QF PCR and
array CGH replacing conventional cytogenetics as the first- line prenatal
testing. Out of 1,049 specimens array CGH was successful in the 99.8%. The
turnaround time was 5.1 days on 98.4% of samples where array CGH was
performed on uncultured CVS or AF. In the remaining samples (4.6%)
culturing was required which delayed the result [51]. Abnormal chromosomes
were found on 156 (14.9%) specimens of which 124 (11.7%) represented
numerical or structural abnormalities. Of those, 104 were identified by QF
PCR and 20 would have been detectable by classical cytogenetics. The

Array CGH in Prenatal Diagnosis

77

remaining 32 cases (3.1%) would have been missed by G- Banding analysis


and were comprised of 13 (1.2%) pathogenic, three (0.2%) VOUS, 10 (0.9%)
benign and six (0.57%) CNVs, known to represent carrier status of a disease.
The study used the targeted array version of 8x60K Agilent array which has
additional probes in the regions of interest for specific to prenatal arrays. This
array however is missing some probes that are applicable to postnatal testing.
The use of such targeted array enabled the group to minimize that incidence of
VOUS. In addition it was demonstrated that pathogenic findings are correlated
with the presence of fetal structural abnormalities but not with increased
nuchal translucency or abnormal first trimester biochemical results.
Srebniak et al. [52] retrospectively reviewed 465 prenatal cases referred
for high resolution prenatal array. They re-evaluated them at 0.3, 0.5 and 1Mb
resolution to assess how many DECIPHER syndromes, out of the 58 listed,
would theoretically have been missed. It was determined that array analysis at
0.5Mb would have missed six DECIPHER syndromes all of which were
considered to be rare syndromes. The number of VOUS also decreases at this
resolution. By evaluating cases in their laboratory at the 0.5Mb resolution they
state that they would theoretically miss two pathogenic CNVs, both of which
were found in cases presenting with ultrasound abnormalities. This group
applies high-resolution analysis in the prenatal cases presenting with
ultrasound abnormalities and they discuss that they would therefore not have
misdiagnosed these two cases as they would not analyze them at the 0.5Mb
resolution. This group has already replaced conventional cytogenetics in
prenatal diagnosis with array CGH and applies high-resolution array CGH in
cases with ultrasound abnormalities. The lower resolution array of 0.5 Mb is
applied for all other referrals [52].
With the application of array CGH on 64 prenatal samples we also aimed
to demonstrate its additional diagnostic value. Array CGH was able to
characterize previous known aberrations in five cases, by determining the
origin of marker chromosomes, as well as delineating a deletion detected in
chromosomal analysis. Array CGH failed to determine the origin of a mosaic
marker found by conventional cytogenetic analysis. The majority of the cases
examined had normal karyotype and ultrasound findings and the remaining
had apparently balanced rearrangements, most of which also had ultrasound
findings. A total of 17 cases appeared to have CNVs (26.5%), four of which
were previously detected by other methods; 13 were from pregnancies with
normal karyotype and ultrasound abnormalities, three of which (4.7%) were
classified as pathogenic. The remaining 10 CNVs were initially categorized as
VOUS and were subsequently re-classified to represent familial CNVs

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

unrelated to the reason for referral. The additional diagnostic capacity of array
CGH in the current study for clinically significant change was determined to
be 10.9% [66].

9.2.2. CNV Classification


By reviewing the publicly available databases, a CNV can be classified as
common or rare. Common CNVs usually represent normal genomic variation
or benign CNVs that are mostly not involved in diseases. In some occurrences
a common CNV can represent a susceptibility locus. CNVs that are rare will
more likely be penetrant for a disease, but some will be benign while other
will still remain of unclear clinical significance [67]. It is important when
comparing CNVs to compare gains with gains and losses with losses as the
potential clinical consequences may differ significantly. The steps followed in
interpreting CNVs are:

Comparison with in-house and international datasets


Comparison with in-house and international affected individual
datasets
Gene content and literature studies

One of the publicly available databases is the Database of Genomic


Variants (DGV http://projects.tcag.ca/variation/) and it provides a useful
register of control data for studies aiming to associate genomic variation with
phenotypic data. Its difference from other databases is that it concentrates
merely on control samples. It is regularly updated with data emerging from
published research studies. High quality studies only are included in this
database. They undergo a series of reviews and only if they fulfil the inclusion
criteria are then imported in DGV.
Variants of greater than 50bp and smaller than 3Mb are included in DGV.
For variants included in DGV a comparison is carried out with the regions
associated with genomic disorders listed on DECIPHER (DatabasE of
Chromosomal Imbalance and Phenotype in Humans using Ensembl
Resources) to ensure that variants in control individuals do not coincide with
known disease-causing variants 67. Once it is determined that a CNV was not
identified in a control set the next step is to determine whether it was
previously found in a patient with similar phenotype. Databases that show
genotype-phenotype correlation exist and are freely available to search from.
Such databases are DECIPHER, ISCA, ECARUCCA. We mainly use the
DECIPHER which is an interactive Web-based database with tools that help

Array CGH in Prenatal Diagnosis

79

us in the interpretation of subtle chromosomal abnormalities. DECIPHER


retrieves information from a variety of resources which are relevant to the
imbalance found in the patient. Known and predicted genes within an
aberration are listed in the DECIPHER patient report, with consent a brief
description of the phenotype is available, genes of clinical importance are
highlighted and common copy- number changes in control populations are
displayed [67].
Finally, once these databases have been consulted, other resources will
need to be searched in order to determine the function of a specific gene that
was included in the aberrant region. A primary resource for connecting genes
to disease related phenotypes, in a general rather than case based manner, is
the Online Mendelian Inheritance in Men database (OMIM [68]). It contains
curated records of genetically inherited human disorders with references to
causative genes or genetic loci.
Deciding to report or not to report a CNV as benign must be done with
caution as it may sometimes contribute to pathogenicity if:

There is a deletion on one allele and a mutated gene on the other allele
[69]
The same deletion is present in both alleles; two benign heterozygote
deletions generating a deleterious homozygous deletion [70]
Each parent has a different benign (heterozygous) deletion in the same
gene, which, when both are inherited there is a deleterious effect on
the offspring (compound heterozygote)
The region contains an imprinted gene with possible difference in
pathogenicity [71]
The CNV is on chromosome X and was inherited by a male offspring
from his unaffected mother [72]
The CNV is inherited from a mosaic carrier, who is not or only mildly
affected [73]
The CNV occurs in combination with another CNV and together these
lead to a pathogenic defect [74]

In these cases any benign CNV will become pathogenic and it must be
reported as such with a detailed explanation in the report.
To summarize, following search in in-house or publicly available
databases CNVs will be categorized as being: 1) pathogenic or likely

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

pathogenic, 2) benign or likely benign, 3) of variable expressivity and


penetrance or 4) coincidental.

9.2.3. CNVs with Variable Expressivity and Penetrance


Several CNVs have been found to be associated with variable penetrance
and expressivity, rendering their classification and possible pathogenic effect
very difficult.
In our clinical setting prenatal array CGH analysis revealed a case with a
maternally inherited duplication of 0.7Mb in size, at the 22q11.2
microdeletion/microduplication syndrome region. This was from a 14 week
pregnancy referred for chromosomal and array CGH analyses due to an
increased nuchal translucency seen on ultrasound. Even though this finding
was reported as likely pathogenic the exact risk for the fetus could not be
estimated as this region is known to have variable expressivity and
intrafamilial variability. This is one of the problems that arise with the use of
array CGH in the prenatal setting; it is difficult to correlate such findings with
the phenotype and this increases the anxiety for the future parents. Like the
22q11.2 microdeletion/microduplication syndrome there are other regions of
variable expressivity and/or intrafamilial variation for instance the 16p11.2
[75], 16p13.11 [76], 7q11.23 [77].
9.2.4. Coincidental Findings
Coincidental findings constitute one of the major challenges in the
implementation of array CGH in the clinical prenatal setting [78-80].
An example of coincidental finding was encountered in our laboratory on
a case initially referred for chromosomal and array CGH analyses because of
absence of the nasal bone on ultrasound screening. Array CGH analysis
revealed three CNVs in total, all of which were inherited from the mother; two
independent duplications on chromosome 9 (0.3Mb and 0.4Mb) and a deletion
of 1.1Mb on chromosome 17 at 17p11.2. The duplications on chromosome 9
were initially of unclear clinical significance, but as they were inherited from
the mother they were considered benign. The deletion on chromosome 17
includes the PMP22 gene (Peripheral Myelin Protein 22) and is consistent
with Hereditary Neuropathy with Liability to Pressure Pulsies (HNPP). This is
a neuropathy with or without symptoms and while this finding was
coincidental and unrelated to the reason for referral it was reported as
causative. Findings such as these ones pose dilemmas to clinicians as they are
hard to deal with. The fact that a CNV is also found in a parent will not always
mean that it is benign. In addition, coincidental findings and their implications

Array CGH in Prenatal Diagnosis

81

are a major issue that needs to be discussed in pre-test counseling extensively.


The patients should be given the choice to choose whether they want to know
or not. Coincidental findings could also occur in cancer predisposition genes.
In a study by Adams et al., out of 18,437 individuals with developmental
disabilities and congenital anomalies tested by array CGH , 34 (0.18%) were
identified as having gains and losses encompassing gene regions associated
with documented genetic conditions of increased risk for cancer [78]. In some
instances this could prove a very powerful prognostic tool for the patient being
tested, especially where early intervention is necessary (identification of RB1
deletion in a one year old child before onset of symptoms). The parents need
to know of the diagnostic capacity of array CGH before testing is completed,
so that they are not faced with an overwhelming reality post- testing. As
Adams et al. continue to discuss, even though most of the cancer
predisposition copy number changes identified by array CGH are de novo
events, they could potentially unmask a familial predisposition to cancer [78].

9.2.5. Pathogenic Findings


Pathogenic findings may be familial or de novo in origin. Below we
describe certain examples from the literature and our own clinical setting
outlining the importance of array-CGH in prenatal diagnosis.
9.2.5.1. Detection of De Novo Pathogenic Findings
We previously reported [16] on a CNV which was found during array
CGH analysis with BAC arrays. A 13 week pregnancy was referred for array
CGH due to a de novo translocation identified during chromosomal analysis. A
de novo copy number loss of 0.2Mb to 1.35Mb in size located at the
translocation breakpoint was detected by array CGH. Confirmation of the
deletion was carried out in the parents as well the fetus by Real Time PCR. At
the time, we reported this finding as having an increased risk for phenotypic
effect in the fetus for three reasons. It was de novo in origin, it was located on
one of the translocation breakpoints and there was an entry with similar
aberration in DECIPHER. It is important to note that the use of BAC arrays
(resolution of 1Mb) did not allow us to estimate the exact size of the deletion
which further generates problems in the interpretation.
Simovich et al. also report on a prenatal case with a de novo apparently
balanced translocation which following array CGH determined to be
unbalanced [81].

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These cases show the importance of testing de novo balanced


translocations by array CGH to potentially lower the phenotypic risk of 6%
[14] found in these cases as discussed by Sismani et al. [15].
Finally, the reliable interpretation of CNV data is part of the array CGH
analysis and reporting. This is a challenging task and one that requires
expertise and knowledge which can be found in various resources. In the
interpretation of the current case in addition to the laboratorys own dataset
several web resources were available and used to guide us through the
complex task of interpreting the CNVs found. It is imperative that these
databases are used to aid in the discrimination between likely pathogenic,
benign or variables of unclear significance. This will assist us further to
correlate the genotype to the phenotype or the ultrasound findings.
Another example of de novo pathogenic finding encountered in our
diagnostic setting and previously reported by us [66], was in a case of a CVS
sample from an 18 week pregnancy referred for chromosomal analysis, due to
increased Nuchal Translucency identified on ultrasound. QF PCR analysis
revealed a normal diploid complement for chromosomes 13, 18, 21 and
normal complement for chromosomes X and Y determining that the fetus was
male. The establishment of cultures for chromosomal analysis failed after
several days of culture. The physician was notified on the 14th day and array
CGH analysis was offered in order to avoid a second invasive procedure to
acquire a new sample for culturing. Array CGH analysis, using 105K
oligonucleotide arrays, revealed a duplication of 2.1 Mb in size on
chromosome 5 and a de novo deletion on chromosome 15 of 2.4Mb in size.
The duplication on chromosome 5 was classified as likely benign as it was
inherited from the healthy father, consequently stressing the necessity of
confirming the presence/ absence of CNVs in the parents to further categorize
them. The deletion on chromosome 15 was reported as likely pathogenic as it
was relatively large in size, it was de novo, the deleted region contained many
genes and was not listed as pathogenic in the publicly available database.
Furthermore, such single segmental imbalances even though they were
determined by array CGH to be de novo, they could be the consequence of the
unbalanced transmission of a derivative chromosome involved in an
insertional balanced translocation (IT) in the parents [21]. Nowakowska et al.
demonstrated that ITs underlie ~ 2.1% of apparently de novo interstitial CNVs.
Such information may not be important to further evaluate the risk for the
current fetus, but it is important for the accurate estimation of the recurrence
risk to family members. Therefore, chromosome visualization after microarray
analysis is essential for delineating the rearrangement and assessing for further

Array CGH in Prenatal Diagnosis

83

potential imbalance (in the immediate or even in the extended family). In the
current case chromosomal analysis carried out in the parents did not detect an
insertional translocation. The deletion was rather small in size to be detected
by chromosomal analysis (2.5Mb), therefore FISH analysis would be have
been necessary to visualize exactly the nature of the imbalance. At the time
FISH analysis was not possible to be carried out therefore a disclaimer was
written on the report regarding this point for the counselor and the couple to
have in mind for future pregnancies.
Finally, the diagnostic benefit of array CGH analysis was again
demonstrated on another prenatal case referred to our center. Amniotic fluid
sample from a 22 week pregnancy was referred for chromosomal and array
CGH analyses due to fetal anomalies detected on ultrasound. The fetus
presented a nuchal fold thickness of 4.2mm, heart problems and flexed-wrists.
Array CGH analysis detected a female profile with a deletion of approximately
2.6Mb in size on the long arm of chromosome 20, on chromosomal band
20q13.13 extending to band 20q13.2 [location 48,456,098-51,041,774 using
build GRCh37 (hg19)]. The deleted region contained many RefSeq genes
including three OMIM disease genes [68] namely Protein-Tyrosine
Phosphatase, Nonreceptor-Type, 1 (PTPN1, OMIM #176885), DolichylPhosphate Mannosyltransferase 1, Catalytic Subunit (DPM1, OMIM #
603503) and the Sal-Like 4 (SALL4, OMIM #607343) (Figure 9.2.5.1).
Following search on the public databases it was determined that the deleted
region was not listed as polymorphic and had overlapping DECIPHER entries
similar size. According to the current literature deletions within the
20q13.13q13.2 region including the SALL4 gene result in the Okihiro
syndrome (OMIM #607323). This syndrome is characterized by upper limb
anomalies indicating that haploinsufficiency of the SALL4 gene is its common
causing mechanism. [68, 82, 83].
Furthermore, deletions including the SALL4 gene and additional genes
within the region, as in the current case, were found in patients featuring
Okihiro syndrome and variable degrees of psychomotor delay. This suggests
that the deletion of additional functional genes in the region results to an
expanded phenotype of Okihiro syndrome plus developmental delay [83]. It
has also been reported that in familial cases of Okihiro Syndrome (affecting
only SALL4 gene) there is significant clinical variability [83, 84]. The parents
were counseled extensively regarding this finding and decided to continue
with the pregnancy. They consented however for further familial investigation.
FISH analysis subsequently carried out showed that the deletion was de novo
in origin and determined the recurrence risk of this finding to be very low. As

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expected, chromosomal analysis could not detect the deletion as it was 2.6 Mb
in size and beyond this assays resolution.
There are many similar examples of de novo pathogenic CNVs in the
literature showing the usefulness of array CGH in prenatal diagnoses [85, 86].

Figure 9.2.5.1. Array CGH analysis of a prenatal case showing a de novo copy number
loss on the long arm of chromosome 20. Representation of the chromosomal and
genomic location region in the Database of Genomic Variants; the G-Banded
chromosome is depicted in the image for comparison. The loss of 2.6Mb in size is
marked by the red arrows, and it encompasses several RefSeq genes (not shown here);
three OMIM disease genes included in the deletion are represented by the orange
bracket PTPN1, DPM1, SALL4.

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85

9.2.5.2. Detection of Pathogenic Findings Originating from Familial


Balanced Rearrangement
Pathogenic findings due to the inheritance of an unbalanced translocation
following the application of array CGH analysis in prenatal cases have been
encountered in our laboratory and are also reported by others [87]. We
previously reported [66] on a 12 week gestational age pregnancy referred for
chromosomal analysis and array CGH due to increased Nuchal translucency
(7.1mm). Chromosomal analysis was normal (46,XY), but array CGH
revealed double segmental imbalance which is usually an indication for the
presence of an unbalanced translocation. Array CGH carried out with 105K
oligonucleotide array showed a terminal deletion of approximately 1.4Mb in
size on the long arm of chromosome 9 and a terminal duplication of
approximately 2Mb in size on the short arm of chromosome 17. FISH analysis,
using subtelomeric specific probes for chromosome 9 and 17, was then
performed and confirmed the array CGH results and determined the presence
of an unbalanced translocation. Retrospective analysis of the fetus karyotype
could not detect the abnormalities, as expected, since the imbalances (1.4Mb
and 2Mb) were beyond the resolution of the karyotype. Chromosomal and
FISH analyses carried out in the parents revealed the presence of a balanced
translocation in the mother between the long arm terminus of chromosome 9
and the short arm terminus of chromosome 17; the translocation was not
visible in the karyotype of the mother. This is a cryptic translocation which
under other circumstances would have been missed. The imbalances found
were likely to be causative and related to the reason for referral as the deleted
region on chromosome 9 overlaps with the 9q subtelomeric deletion region
and included many genes several of which were OMIM genes. In addition, the
duplicated region on chromosome 17 contained many genes, including two
OMIM genes, and partially overlapped with the Miller-Dieker syndrome
region. The couple went through counseling for further explanation of the
implications of the findings for the current pregnancy, as well as, for future
pregnancies. They elected to terminate the pregnancy. The importance of
Genetic Counseling is further discussed in the specific subsection.
The usefulness of the additional information array CGH provided in the
diagnosis in this case is obvious. Without it, this double imbalance would have
remained undetected. Furthermore the information acquired from this case will
be used by the family for the better management of their pregnancies in the
future. After careful evaluation of this couples reproductive and medical
history, it was revealed that they had a previous pregnancy which was
terminated due to multiple, severe ultrasound findings (Tetralogy of Fallot,

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talipes and other). The couple also had an affected child. Both the previous
pregnancy and the affected child were investigated in the pre-array CGH era
by our laboratory and showed normal karyotypes; retrospective G- Banding
analysis did not detect the abnormalities, and the parents consented to perform
array CGH on stored genetic material. Array CGH analysis revealed related
findings to the current case and contributed to the diagnosis for their affected
child. The importance of having the pedigree of a family being investigated is
paramount as shown in this case. Had the parents informed the clinicians
during the previous pregnancy that they already had an affected child the
management of the first pregnancy might have been different. The first
pregnancy was investigated by chromosomal analysis on Amniotic Fluid
sample on the 16th week and revealed normal karyotype. It was terminated
based on the ultrasound findings despite the fact that the karyotype was
apparently normal. Had the parents known at the time that their born child had
an inherited chromosomal abnormality, they would have opted for an earlier
prenatal diagnosis on their first pregnancy perhaps by chorionic villus
sampling. This would have lessened their anxiety.

9.2.6. Copy Number Variations (CNVs) Polymorphisms


In a study carried out by our group (unpublished data) a different approach
was used, on a research basis, with samples which had a normal karyotype but
presented ultrasound findings. In an attempt to map copy number variations of
the Cypriot population, a single Male reference DNA was used as our control
DNA, as opposed to pooled male/female control DNA used in the other array
CGH analyses. The main idea was to build a database which would contain all
this information to be used later on as a reference for analysis. The huge
amount of CNVs picked up in the analysis of this group, most of which turned
out to be benign/ common polymorphisms shared in the population, could not
be directly compared to the other groups. There were only two CNVs which
could be possibly pathogenic but more cases with the same ultrasound findings
have to be studied in order to classify these findings as being causative.
9.2.7. New Microdeletion Syndromes That Emerged from High
Resolution Array CGH Analysis
Over the years array CGH has contributed to the characterization of new
microdeletion/ microduplication syndromes by screening large patient cohorts
with intellectual disabilities. This is a benefit as diagnosis was provided to
even more patients with intellectual disabilities. Even more of these novel
syndromes may be identified in the future with the obvious value it will offer

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87

to the medical society. Array has facilitated a reverse dysmorphology


approach in contrast to the earlier phenotype first approach. What this means
is that the array results from a large cohort of patients are used to define a
critical chromosome region that is deleted/duplicated in several patients.
This is followed by comparison and study of the clinical features of the
patients, to determine the essential phenotypic findings. Then if the clinical
phenotype is distinctive enough searching for other patients who havent been
screened, with similar features could identify others with the same abnormality
[88]. Examples of such syndromes are the:

15q13.3 deletion syndrome


16p11.2 deletion
16p11p12.1 deletion syndrome
2p15p16.1 deletion syndrome
15q24deletion syndrome
1q41q42 deletion syndrome
9q22.3 deletion syndrome

The choice of the array type to be used is critical in order to be able to


uncover new microdeletion/microduplication syndromes. The use of targeted
arrays for example could minimize the chance of novel syndromes to be
identified. These arrays examine loci of known clinical significance in order to
reduce the probability to detect CNVs of unclear significance. On the contrary,
the use of whole genome arrays offers more chances of previously undetected
aberrations to be discovered, even though this is not always a straight forward
answer as there is a higher detection rate of CNVs with whole genome arrays.
Targeted arrays, though, with enriched probes to potential hotspots in
the human genome associated with rearrangements, could lead to the
identification of novel deletion/duplication syndromes. Examples of such
hotspots are segmental duplications located in certain chromosomes.

10. PRE AND POST-TEST GENETIC COUNSELING


As genome-wide analysis is being introduced into prenatal diagnosis, pretest counseling is of paramount importance due to the nature of the test and the
findings that may emerge from the analysis. Counselors need to be explicit and

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offer information in a non-directive way, so that prospective parents can


decide on their own, having their future childs best interest in mind.
It is very crucial that the prospective parents provide clinicians with their
own medical history, the medical history of the pregnancy including
ultrasound findings and the family pedigree of both parents up to three
generations.
Counselors should be aware of the state of mind parents-tobe are in, right
after an ultrasound abnormality has been detected. Parents may not be able to
absorb any information given to them at the time, so it is good practice to have
everything written down as well so that it is available for them to read later on.
Following this, parental consent should be obtained. Prospective parents
should be informed of the test and its limitations. They should be made aware
that the array technique cannot detect all diseases or well known syndromes.
In a study of 141 fetuses with ultrasound abnormalities and normal array
results, there was a diagnosis in 15% of them when they were reviewed
postnatally [80].
If, in the course of testing the fetus, whole-genome array analysis is
needed to be carried out for the parents, they should be counseled
appropriately including informed consent on what information they want to
receive.
The parents should be aware of all the possible outcomes of the array
testing which could either be normal or abnormal. It should be explained to
them that if CNVs are detected they could either explain the fetal ultrasound
abnormalities or be de novo and of unknown clinical significance. In addition,
if CNVs are found they could also have been inherited from either of the
parents and be of unknown clinical significance. Finally they may represent an
unsolicited finding unrelated to the ultrasound findings.
Variables of unknown significance (VOUS) and incidental findings are
the most challenging for counselors. This is why it is of prime importance to
inform parents of such possible findings. An example is a late-onset inherited
disease either de novo or inherited in the family. Its implications should be
explained and a distinction should be made between treatable (hereditary
cancer) and non-treatable (Huntingtons disease) late-on-set diseases. There is
no straight forward guideline on how this should be carried out. For instance,
in Europe the current tendency is to ask parents whether they want to be
informed about treatable late-onset diseases. Some laboratories even have a
policy of not reporting unsolicited CNVs to non-treatable diseases [80]. There
are many ethical questions arising from all these, one of them being the extent
to which pregnant women and their partners should be allowed to determine

Array CGH in Prenatal Diagnosis

89

the range of possible outcomes that will or will not be reported back to
them [89].
Faas et al. argue against the increase of VOUS anticipated with the nontargeted whole genome approach compared with karyotyping, as this was not
confirmed by their results [90]. Srebniak et al. report, based on unpublished
data,that even though they do not report VOUS themselves [52, 91], most of
the VOUS found in their clinical setting were inherited from a parent (80%)
and could potentially classified as benign.
National guidelines in the use of array CGH in prenatal diagnosis remain
to be established.

11. DETECTION OF ANEUPLOIDIES/CNVS IN


POC/INTRAUTERINE DEATH/STILLBIRTH SAMPLES
Of all the recognized pregnancies, about 10-15% ends in clinical
miscarriage or spontaneous abortion, usually towards the end of the first
trimester. Out of these about 50% are shown to have a chromosomal
abnormality, if they are all successfully cultured [1, 2]. It has been
demonstrated that the success rate in these samples is about 75% [92, 93].
Fritz et al. suggest an even higher aneuploidy rate (72%) in specimens that
failed to grow in vitro and were analyzed with metaphase CGH [2]. The
advantage of these assays is that they circumvent technical problems
associated with tissue culturing.
In a study of first trimester fetuses that failed to grow in vitro analyzed
with 1Mb BAC array [94], 15 out of the 26 POC samples had abnormal
profiles (57.7%), 13 of those being chromosomal aneuploidies (86.6%). The
remaining two had a single clone deleted in one and a single clone duplicated
in the other. Based on the calling criteria where, for BAC arrays, two
consecutive clones have to deviate in order to be called a CNV, these two
cases couldnt be considered abnormal unless further testing was carried out.
The same study also noted the detection of autosomal monosomies, a finding
that is rarely detected in cultured POC samples. The most likely explanation
for this being the fact that specimens containing these chromosomal
abnormalities do not do well when cultured and fail to produce analyzable
metaphases for conventional cytogenetics. This could further explain the
failure of some samples to grow in vitro [93, 94].

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In our experience most of the abnormalities detected within this group of


samples that do not grow in culture and are analyzed with other molecular
techniques, such as QF PCR or array CGH, are very similar to those found in
POCs that grow in culture. Most of these however represent pregnancies that
miscarried earlier in pregnancy and did not reach the point where there could
be obvious ultrasound findings. For such cases, arrays with higher resolution
do not offer additional diagnostic information [95]. The higher costs and the
possibilities of unsolicited findings during the analysis of these cases do not
make high resolution arrays an appealing application. There are commercial
arrays from some companies which are more suitable for these types of
samples as they have lower resolution to serve the purpose of the analysis. Bug
et al. suggest on a CGH+SNP platform that reliably detects aneuploidy and
Uniparental Isodisomy in one assay [95]. In this pilot study it is demonstrated
that segmental aberrations in POC samples can be detected without the
excessive calling of micro- alterations which if found may need to be
classified [95].
Studies where the application of array CGH was on fetuses with multiple
malformations appear to have different results as compared to the fetuses with
no ultrasound findings. There are several examples in the literature showing
that the detection rate in microdeletions/microduplications is higher in those
samples that were presented with a number of serious ultrasound findings. In a
study of 49 fetuses with multiple malformations and normal karyotype,
targeted BAC array was used and a detection rate of 8% (4/49) causative
imbalances was reported. [61]. Another group applied array CGH
retrospectively in 50 fetuses with multiple malformations using a 44,000
oligonucleotide array and identified causative imbalances in 10% (5/50) [96].
Vialard et al. demonstrated a 10.8% detection rate by performing array CGH
on 39 consecutive fetuses with multiple congenital abnormalities; 37 had
normal karyotype and 2 had a de novo unbalanced karyotype. Targeted BAC
array successfully further characterized the two abnormalities detected by
cytogenetic analysis and detected another four abnormalities (4/37, 10.8%)
[59].
Finally in a study where whole-genome array CGH was applied on fetuses
presenting with at least one malformation detected on ultrasound, but for
whom standard genetic analyses failed to provide a diagnosis, showed
clinically significant aberrations in 8.2% of tested fetuses. It also showed
unclear clinical significant results in 12.2% of the tested subjects [97]. Table
11 shows the comparison between these studies.

Array CGH in Prenatal Diagnosis

91

These data support and suggest the implementation of array CGH, as its
application offers additional diagnostic information in as much as 10% of
cases were the fetuses have malformations and a normal karyotype. However
it also presents us with the problem arising with variables of unclear
significance (VOUS). Wang et al. [93] report on the application of Oligo SNP
CMA array on 268 POC specimens. Included in these were 32 specimens
which were also analyzed with G- Banding and 107 which had culture failures.
The remaining 129 were only analyzed by arrays. The study set different
thresholds for gains, losses and regions of homozygosity which were
determined to be >200kb, >50kb and >10Mb respectively. In the group of
samples where both methodologies were applied the concordance in the results
was 81% with five abnormalities missed by G- Banding. Those abnormalities
were one cryptic unbalanced translocation and four cases of VOUS. In
addition one case which was reported as normal by array CGH appeared to
have an apparently balanced translocation in G- Banding analysis. The overall
abnormality rate of specimens studied by Oligo-SNP CMA was 44.6%. In
addition two cases with segmental UPD were also observed.
Finally, Bug et al. also demonstrate the usefulness of a CGH+SNP 8x60K
array for the detection of absence of heterozygosity in one POC sample and
the formation of complete hydatidiform in one patient [95]. The frequencies of
UPD in POC are reported by some studies as low, but it is discussed that there
may be an underestimated pathogenic factor for these types of samples
because of the limits the current detection methods have [98-100].
In our laboratory QF PCR is initially applied in this type of samples to
exclude the most common aneuploidies observed in first trimester
pregnancies; in those that are normal array CGH is applied. The benefits these
methods (QF PCR and array CGH) offer, in POC/intrauterine death/stillbirth
samples are evident considering the fact that around 30% of the total of these
samples received by the laboratory over a year would have failed and no
results would reach the patients. Moreover, the turnaround time for reporting
is dramatically decreased when it is compared to G- Banding. Finally, a very
small amount of DNA is required for both of the analyses to be carried out.
Wang et al. also comment on the enhanced success rate of 90% versus 75% of
array CGH analysis over conventional cytogenetics and of the lower
turnaround time [93]. The limitations of DNA- based methods such as these lie
with the fact that QF PCR is not a genome-wide analysis method and array
CGH cannot detect balanced rearrangements and triploidies.

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Table 11. Comparison between studies which used array CGH in
POC/Intrauterine Death or Stillbirth Samples

Study

Array Type

Benkhalifa
et al., 2005
[94]
LeCaignec
et al., 2005
[61]

1 Mb BAC/PAC
targeted array

Karyotype/ Reason
for carrying out
array CGH
Unknown/ Failure
to grow in vitro

BAC/PAC Targeted
array

Normal/ Multiple
Malformations

5/49
Abnormal

Valduga et al.,
2010 [96]
D'Amours
et al., 2012
[97]

44,000
Oligonucleotide array
Whole genome array
(BAC, 105K and
135K oligonucleotide
arrays)

Normal/ Multiple
Malformations
Normal Karyotype/
At Least one
malformation

5/50
Abnormal
10/49
Abnormal

Wang et al.,
2014 [93]

Oligo SNP CMA


2.67M (1.9M copy
number +750K SNP
probes)

Normal karyotype
or Failure to grow
in vitro

107/240
Abnormal

Results
15/26
Abnormal

Clinical
Significance
of Results
57.7%
Causative
8.2%
Causative, 2%
Unclear
significance
10%
Causative
8,2%
Causative,
12,2%
Unclear
Significance
44.6%
Causative

12. CAN ARRAY CGH ANALYSIS FULLY REPLACE


KARYOTYPING?
Array CGH analysis is being introduced in prenatal diagnosis in
conjunction to chromosomal analysis, but it has not yet fully replaced
karyotyping for the following reasons:
a) Balanced rearrangements such as translocations, balanced insertions
and inversions cannot be detected. This is especially important in
Robertsonian translocations as carriers of such are at high risk for
uniparental disomy [101], and the risks UPD implies as they were
discussed previously. Even in the case were SNP arrays are used
which can detect isodisomy [102] they cannot detect heterodisomy
which is the most common form of UPD. In addition to Robertsonian
translocation, balanced rearrangements especially de novo reciprocal
translocations or insertions are important to be detected as they can
sometimes lead to abnormal phenotypes for the reasons previously
mentioned. Furthermore the awareness of the presence of a balanced

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93

rearrangement can provide the couple future risk assessments for an


unbalanced offspring and information useful for reproductive
planning
b) Low level mosaicism often seen in prenatal diagnosis cannot be
detected. Mosaicism is detected in 1-2 % of CVS samples and in 0.2%
of amniotic fluid samples [9,103]. Even though in about 84% of
mosaic cases in CVS, the mosaicism is confined to the placenta [104],
the remaining cases could remain undetected if array CGH is the only
method applied
c) The presence of marker chromosomes even in the non- mosaic state
cannot always be detected. Marker chromosomes are encountered in
about 0.1% of prenatal diagnoses [103] and very often in the mosaic
form. Depending on which chromosome they are derived from, their
size, their inheritance mode and whether they are euchromatic or
heterochromatic the phenotypic risk can be determined. In a study of
55 cases with marker chromosome it was demonstrated that out of the
26 non-mosaic markers only 14 were detected leaving 46% of array
results normal. Even if this percentage reflects that the markers are
mainly heterochromatic, the lack of detection does not completely
exclude a possible phenotypic effect [105]
d) The type of rearrangement cannot be visualized in the event were
deletion or duplication detected by array CGH is proven to be de novo
after parental tesing.

13. FUTURE APPROACHES IN PRENATAL DIAGNOSIS


13.1. New Approaches in Non- Invasive Testing
The introduction of Non-Invasive Prenatal Testing (NIPT) will probably
overcome the problem of who should be screened or not; whether it will be all
pregnant women or those at high risk. NIPT might replace all current
biochemical screening tests or be the first-tier screening test after an indication
of Down syndrome by a biochemical screening [106].
NIPT for Down syndrome is rapidly evolving. Recent research shows that
trisomy 21 can be reliably determined from the analysis of cell-free fetal DNA
from maternal plasma [107,108]. Until today two methodologies have
accomplished the development of NIPT methods for Down syndrome with
positive results. They are the next- generation sequencing technologies [109]

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Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani

and the Methylation Dependent ImmunoPrecipitation (MeDIP) real time


quantitative PCR based approach [110].
The next-generation method can analyze the nucleotide sequences of
millions to billions of DNA molecules in one run, being able to identify and
count the frequency distribution of DNA molecules in a sample. Based on the
fact that maternal plasma DNA could be sequenced to identify the
chromosomal origin of each DNA molecule, the proportion of molecules from
a potentially aneuploid chromosome (for example chromosome 21) could be
determined. Based on this, Lo et al. [111] demonstrated that the proportion of
chromosome 21 DNA molecules were elevated, in plasma of pregnant women
carrying a trisomy 21 fetus compared to that of euploid pregnancies [109].
This approach is highly accurate and very promising, as proven by two groups
[109,112], for the direct detection of trisomy 21. The only drawback for this
method is the fact that it is high cost and low throughput, only a small number
of cases can be analyzed simultaneously and the results take several days to be
available.
The MeDIP real time quantitative PCR methodology is built on the fact
that there are differences in methylation between the mother and the fetus.
Papageorgiou et al. [113] developed a method which was based on the
investigation of fetal specific methylation markers using the methylated DNA
Immunoprecipitation methodology in combination with Real Time
quantitative PCR. In the first trials of the method it provided 100% sensitivity
and specificity. The MeDIP real time quantitative PCR methodology is a new,
fast, and cost- effective NIPD for Down syndrome that can be offered as early
as the 10th week of gestation. Once the larger scale validation study is
completed (700-1000 samples) this method can be used in clinical practice
[114]. The field of NIPT has evolved and is offered for aneuploidies for other
chromosomes and in the future it may even be offered for small
rearrangements. Further studies are needed, to establish whether it could,
completely replace invasive prenatal diagnosis methods.
With the use of NIPT caution should be applied when interpreting the
results as the fetal cffDNA circulating in maternal blood is originating from
trophoblast and consists of DNA from the placenta [115,116], leading to a
discordance between the NIPT result and the actual genetic constitution of the
fetus [117]. Dugo et al. suggested that the results of NIPT tests should be
confirmed by conventional cytogenetics or array CGH [118]. In their center
there were six consecutive false positive cases from cffDNA testing; three
involving sex chromosome abnormalities and two involving autosomes (13,
18). All were found to be normal following invasive prenatal diagnosis. To the

Array CGH in Prenatal Diagnosis

95

best of our knowledge, as of today, two groups have identified false negative
cases. Smith el al. report on a pregnancy testing negative for Trisomy 21 by
NIPT, leading to the birth of a Down syndrome child [119]. In a study of 3000
consecutive pregnancies in Belgium and The Netherlands two false negative
results were reported, one trisomy 21 and one trisomy 18 [120].

13.2. Next Generation Sequencing and Prenatal Diagnosis


Deep sequencing of the whole genome is a promising methodology, but is
not currently routinely applied in invasive prenatal diagnosis. Talkowski et al.
report on the retrospective application of massively parallel paired end
sequencing of customized large-insert jumping libraries. They aimed to
precisely identify the breakpoints of an apparently balanced translocation
46,XY,t(6;8)(q13;q12.2)dn detected by conventional Cytogenetics in a
prenatal case [121], and to further demonstrate the disruption of genes at the
translocation breakpoints. The fetus presented with an isolated heart defect at
18.8 weeks of gestation along with some other abnormalities in other organs.
Ultrasound findings increased with increasing gestational age and due to
absence of fetal movement at 36.2 weeks an emergency caesarean section was
performed. On delivery a diagnosis of CHARGE syndrome (OMIM # 214800)
was made based on the clinical features of the infant. Its clinical condition
worsened to finally die at 10 days of age. Sequencing proved a valuable tool in
defining the exact breakpoints of the translocation and determined that the
CHD7 gene located at one of the translocation breakpoints (8q12.2) was
disrupted; CHD7 is a pathogenic locus for CHARGE syndrome [122].
Sequencing was therefore able to provide a consistent diagnosis with the one
made on the clinical findings on postnatal examination of the infant [121].

CONCLUSION
Karyotyping has been the golden standard method for prenatal diagnosis
for decades, being able to sufficiently diagnose numerical and large structural
abnormalities (<3-10Mb). With the introduction of array CGH analysis in
postnatal analysis and its use as a first-tier test in cases of intellectual
disabilities, it has been postulated that this method might someday actually
replace conventional cytogenetics in prenatal diagnosis as well. Array CGH in
a postnatal setting, has been demonstrated to be a high throughput,

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comprehensive and fast in detecting copy number changes that can go


undetected by light microscopy. It has been demonstrated throughout this
chapter that in order for array CGH analysis to reach its full diagnostic
capacity in prenatal diagnosis, the appropriate platform and reference DNA
should be selected. More importantly, it has clearly shown through several
examples presented in this chapter that array CGH is a valuable tool in
prenatal diagnosis, both in cases with fetal malformations and normal
karyotype, as well as in cases were an abnormality was detected with another
method and further investigated with array CGH. Array CGH provided
valuable information for phenotype-genotype correlation and provided more
accurate information regarding the clinical significance and the risk in the
current and future pregnancies of the respective patient. Another critical factor
for accurate CNV classification is parental testing to determine between
familial and de novo CNVs. Appropriate pre and post- test genetic counceling
offer the prospective parents tools to decide on the management of their
pregnancy. However, one of the problems posing dilemmas to genetics
councelors and something that array CGH has to overcome is the fact that it
can detect coincidental findings, variants of unknown significance as well as
variants with variable expressivity. Furthermore, array CGH could be used in
POC/intrauterine death/stillbirths samples were malformations exist in the
fetuses, using the same platform as in prenatal cases, as it offers an increase in
detection rate in this category of samples. Array CGH can also be applied in
samples were there are no ultrasound findings in the fetus, after they have been
analyzed with QF PCR to exclude common aneuploidies. For this category of
samples lower resolution arrays could be used. Currently the ideal setting to
advance prenatal diagnosis and increase its resolution is the application of
array CGH in high risk pregnancies in conjunction with chromosomal analysis
with a microarray designed especially for prenatal diagnosis. As we have seen
this increases the detection rate for likely pathogenic CNVs up to 5%. To
avoid interpretation problems (previously discussed) these arrays could only
cover all known pathogenic CNVs and have a low resolution backbone for
the detection of relatively large CNVs thus keeping the detection of CNVs of
unclear significance to the minimum. A shared database specifically dedicated
to prenatal diagnosis coupled with the growing amount of data regarding
CNVs and dosage sensitive genes could make it easier to interpret genomic
arrays.

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97

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INDEX
#
3D images, 25

A
abuse, 48
acetylcholine, 11, 13
acid, viii, 2, 70
acrocentric chromosome, 62
addictive, viii, 1
adrenal hyperplasia, 43
adults, viii, 7, 8
advancement(s), vii, x, 41
adverse effects, vii, viii, 2, 9
aesthetic, 18
African American women, 15
age, 9, 22, 23, 25, 47, 50, 56, 74, 75, 76, 95,
98, 106
agonist, 11
alcohol consumption, 15
allele, 43, 45, 79
alters, 3, 13
alveolar ridge, 22, 23, 25, 26, 27, 29, 36
alveoli, 3
alveolus, 18, 21, 22, 25, 36
ammonia, 10
amniocentesis, x, 41, 48, 58, 59
amnion, 58
amniotic fluid, viii, 2, 55, 58, 66, 93

amplitude, 11
anatomy, 19, 27, 30
aneuploid, 94
aneuploidy, 28, 31, 44, 48, 50, 57, 67, 68,
89, 90, 97, 99, 104, 106
antigen, 43
anxiety, 70, 74, 75, 76, 80, 86
apnea, 11
apoptosis, 42
arousal, ix, 8, 11, 13, 14
arteries, 3
arterioles, 3
Asian countries, 19
assessment, 23, 25, 26, 33, 34, 35, 36, 38,
51, 72
asthma, 6
asymptomatic, 72
audit, 30
autism, x, 53
awareness, 12, 93

B
babbling, 9, 15
BAC, 69, 70, 71, 73, 74, 75, 76, 81, 89, 90,
92
base, 29, 43, 44
base pair, 44
basement membrane, 2
behavioral problems, 103

108

Index

Belgium, 95, 107


benefits, 13, 57, 91
benign, 65, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 87, 89, 98, 103
biochemistry, 57, 61
biological processes, 59
biosynthesis, 11, 12
birth rate, 20
birth weight, 3, 20
births, 18, 19, 22, 65, 67
bleeding, vii, viii, 2
blood, viii, 1, 2, 43, 45, 48, 51, 55, 59, 73,
95, 106
blood flow, viii, 2, 3
blood group, 43
blood pressure, viii, 2
bloodstream, 2
bone(s), 32, 34, 56, 61, 81
bone growth, 34
bradycardia, 61
brain, viii, 1, 10, 11, 12, 13, 15, 16, 32, 38,
100
brain abnormalities, 100
brain growth, 12
brain structure, 10
Brazil, 35
breathing, ix, 7, 11, 13, 14

C
caesarean section, 95
calcification, 2
cancer, 69, 81, 89, 103
carbon monoxide, viii, 1, 10
catecholamines, viii, 2, 3
cDNA, 71
cell line, 65, 105
central nervous system (CNS), viii, 2, 13,
15, 32, 33, 73
cervix, 58, 73
challenges, xi, 12, 54, 72, 80, 101, 102
chemicals, viii, 1, 10
child development, 14
childhood, 44, 50, 55

children, x, 6, 14, 15, 16, 18, 21, 34, 53, 64,


98, 100
China, 41
chorion, 97
chorionic villi, 58, 60
chorionic villus sampling, x, 41, 42, 86
chromosomal abnormalities, 35, 55, 68, 79,
90, 101, 104
chromosome, 43, 44, 46, 50, 58, 60, 61, 62,
63, 65, 67, 68, 69, 70, 74, 80, 81, 82, 83,
84, 85, 87, 93, 94, 97, 98, 99, 100, 101,
103, 104, 105
chromosome 10, 74
chronic obstructive pulmonary disease, 6
cigarette smoke, viii, 6, 7, 8
cigarette smoking, vii, viii, 6, 7, 8, 12, 13,
14, 15, 16
circulation, 2, 10, 73
classification, x, 22, 33, 54, 72, 80, 96
cleft lip, ix, 3, 17, 19, 20, 21, 22, 23, 25, 26,
27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39
cleft palate, ix, 3, 17, 19, 20, 22, 23, 25, 26,
28, 31, 32, 36, 37, 38, 104
clinical diagnosis, 100
clinical disorders, 67
clone, 90
cognitive development, vii, viii, 7, 8
colic, 9, 13
Comparative Genomic Hybridization
(CGH), v, vii, x, xi, 53, 54, 55, 58, 62,
64, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 89,
90, 91, 92, 93, 95, 96, 97, 98, 100, 101,
102, 103, 104, 105
complement, 18, 38, 55, 65, 82
complexity, 98
complications, 3, 5, 42, 57, 59
conception, 59, 60
concordance, 91
congenital adrenal hyperplasia, 49
congenital anomalies, x, 18, 53, 62, 81, 100
congenital malformations, 19
congenital structural abnormalities, ix, 17
consent, 79, 104

109

Index
construction, 46
consumption, vii, viii, 1, 2, 3, 4
contamination, 60, 68
controversies, 105
correlation, x, 54, 66, 79, 96
corticotropin, 50
cost, 25, 48, 94
cotinine, 4, 14
counseling, 25, 70, 72, 81, 86, 88
covering, 69
culture, 60, 82, 90, 91, 97
cyst, 61
cystic fibrosis, 55, 57
cytogenetics, xi, 54, 62, 63, 64, 67, 69, 70,
71, 73, 74, 77, 90, 92, 95, 96, 106
cytomegalovirus, 57

D
database, xi, 35, 54, 79, 83, 86, 97
deaths, 42
defects, ix, 3, 17, 18, 19, 21, 27, 56, 58, 71,
103
deficiency, viii, 2, 103
deficit, viii, 2
deoxyribonucleic acid, 10
deposition, 2
detectable, 77
detection, ix, xi, 17, 18, 20, 21, 23, 25, 26,
31, 32, 33, 34, 35, 36, 43, 44, 45, 48, 50,
51, 54, 55, 66, 68, 70, 73, 75, 87, 90, 91,
93, 94, 96, 99, 100, 101, 102, 103, 104,
105
developed countries, vii, 1
diploid, 82
disability, x, 53, 55, 62, 64, 67, 103
discordance, 95
discrimination, 49, 82
disease gene, 83, 84
diseases, x, 41, 42, 43, 44, 50, 78, 88, 89
disorder, 43, 55, 57
disposition, ix, 8, 10
dissociation, 58
distribution, 13, 20, 98
diversity, 22, 47

DNA, v, 10, 14, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 58, 59, 60, 62, 67, 68, 69,
70, 71, 73, 86, 92, 94, 95, 96, 101, 105,
106
DNA sequencing, 50, 51
DNA testing, 106
dopamine, 11
dopaminergic, 11
dosage, xi, 54, 62, 69, 97
Down syndrome, 42, 94, 95, 106
drugs, 6
dysmorphism, x, 53

E
electroencephalogram, 8, 13
emergency, 95
endocrine, 3
environmental tobacco, 6
epidemic, vii, 1
epidemiology, 6, 36
epigenetic modification, 16
epigenetics, 14
epiglottis, 26
epinephrine, 11
ethical issues, xi, 54
ethics, 104
euploid, 44, 94, 98
Europe, 19, 89
evoked potential, 9, 11, 14, 15
examinations, 20, 23
excitation, 9
exposure, ix, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14,
15
expressivity, 80

F
false negative, 95
false positive, 22, 23, 25, 29, 31, 95, 106
families, 57
family history, 21, 31, 74, 75, 76
family members, 83
fetal abnormalities, 33

110

Index

fetal development, 2, 42, 59


fetal growth, 5, 56
fetus, vii, viii, xi, 1, 2, 3, 4, 27, 43, 44, 45,
46, 47, 48, 50, 54, 55, 56, 58, 59, 60, 80,
82, 83, 85, 88, 94, 95, 96, 103, 104
fibrin, 2
fibroblasts, 58
Finland, 21, 36
fish, 99
fluid, 27, 83
fluorescence, 68, 69
forebrain, 10
formation, 2, 11, 91
fragments, 42, 44, 69
frequency distribution, 94

G
gene expression, 13
gene regulation, 15, 99
genes, xi, 10, 43, 44, 48, 54, 62, 63, 67, 71,
79, 81, 83, 84, 85, 95, 97, 104
genetic counselling, 104
genetic disease(s), vii, x, 41, 42, 43
genetic disorders, x, 42, 43, 44, 53, 55, 69
genetic information, 47, 48
genetic syndromes, ix, 18, 19
genetic testing, 106
genetics, 96, 97, 103, 106
genome, 43, 44, 45, 46, 47, 50, 55, 66, 69,
70, 71, 73, 74, 75, 76, 87, 88, 89, 91, 92,
95, 98, 101, 102, 104, 105, 107
genomic regions, 45
genotype, x, 54, 59, 66, 79, 82, 96, 103
genotyping, 49, 74
gestation, 2, 3, 10, 11, 21, 27, 30, 32, 33, 38,
42, 58, 59, 94, 95
gestational age, 20, 22, 23, 25, 30, 32, 34,
58, 60, 61, 85, 95
gestational diabetes, 5
growth, 10, 34
guidance, 58, 59
guidelines, xi, 54, 89

H
haploid, 46
haplotypes, 45, 46, 47
healing, 34
health, viii, 1, 4, 6, 10
heart rate, vii, viii, 2
heterozygote, 79
high blood pressure, vii, viii, 2
high-risk populations, 22
high-risk women, 23
history, 8, 57, 88
Hong Kong, 17, 35, 38
hormone, 50
host, 62
hotspots, 88
human brain, 11, 13
human genome, 44, 69, 72, 88, 99, 100
human health, 48
hybridization, 69, 71, 98, 99, 100, 101, 102,
104
hydatidiform mole, 65, 99
hydrogen cyanide, 10
hydrops, 73
hypertension, 5
hypertrophy, 2

I
identification, 55, 62, 81, 88, 101
image(s), 23, 25, 26, 29, 30, 31, 37, 71, 84
imbalances, 62, 70, 73, 83, 85, 90, 98, 102
impairments, 9
imprinting, 62, 65, 103
in situ hybridization, 69, 98
in utero, 10, 38
in vitro, 60, 89, 92, 104
incidence, 19, 36, 67, 77, 105
incompatibility, 43
individuals, 55, 64, 69, 70, 71, 79, 81, 100,
103
infancy, 10, 42
infant behavior, vii
infant care, 35

111

Index
infants, ix, 8, 9, 11, 12, 13, 14
informed consent, 88
ingredients, 10
inheritance, 65, 85, 93
injections, 16
insertion, 59, 63
integrity, ix, 7, 12, 30
intellectual disabilities, vii, 87, 96
intelligence, 64
interphase, 67
intervention, 81
intrauterine growth retardation, 6
inversion, 63
isochromosome, 65
Israel, 19

J
jumping, 95

K
karyotype, xi, 18, 54, 55, 58, 59, 63, 66, 69,
73, 75, 76, 78, 85, 86, 90, 91, 92, 96,
102, 105
karyotyping, x, 53, 59, 71, 75, 89, 92, 96,
99, 102, 104, 105
Korea, 30, 31

L
labeling, 69
Latin America, 19
lead, 25, 55, 80, 88, 93
learning, 25, 32, 34
lesions, 3
life cycle, 4
light, x, 2, 53, 96
loci, 70, 71, 79, 87
locus, 63, 67, 78, 96
low risk, 23, 31, 35, 101
lower lip, 27
Luo, 100
lymphocytes, 59

M
magnetic resonance (MR), ix, 18, 19, 32,
38, 39, 103, 105
magnetic resonance imaging (MRI), ix, 18,
19, 32, 33, 34, 38, 39, 56
management, vii, x, 18, 37, 54, 57, 86, 96
mandible, 23, 27
manipulation, 23, 26
mapping, 45
marijuana, 13
mass spectrometry, 43
maternal cigarette smoking, vii, viii, 7, 8, 14
maternal circulating nucleic acid, vii, x, 41
maternal smoking, vii, viii, 6, 7, 8, 9, 10, 11,
12
maxilla, 27, 30
measurement(s), 3, 13, 48, 50, 56
media, 3
median, 8, 20, 25, 26
medical, 59, 86, 87, 88, 97
medical history, 86, 88
medicine, ix, 18, 26, 106
medulla, viii, 2
meiosis, 46, 61, 62
membranes, vii, viii, 2, 60
mental retardation, 71, 97, 98, 100
meta-analysis, 49, 102
metabolism, 2
metaphase, 58, 66, 69, 71, 89, 102
methodology, 31, 68, 72, 94, 95
methylation, 10, 14, 49, 94, 105, 106
mice, 11
microRNA, 10
microscope, 58, 69
microscopy, x, 53, 96
miscarriage(s), x, 41, 42, 55, 56, 58, 60, 62,
64, 75, 89
mitosis, 59
modifications, 10
molecular cytogenetics, 66, 67
molecules, 42, 94
monosomy, 60, 100
morbidity, 18
morphological abnormalities, ix, 3, 17

112

Index

morphology, 25, 32
mortality, vii, 1, 3, 18
mosaic, 60, 74, 78, 80, 93
mRNA, 43, 44, 50
mucus, 101
mutant, 43
mutation(s), x, 41, 43, 47, 50, 71, 98, 104,
107
mutation rate, 47, 98

N
necrosis, 42
nervous system, 9
Netherlands, 20, 25, 95, 107
neurological abnormalities, x, 53
neurons, 10
neuropathy, 81
neurotransmission, 11
neurotransmitter(s), ix, 8, 10, 11
newborns, vii, ix, 7, 8, 20, 65
nicotine, viii, 1, 2, 3, 7, 8, 10, 12, 13, 15, 16
nitrogen, 10
nonsmokers, viii, 2
Norway, 20, 36
nuclei, 67
nucleic acid, vii, x, 41, 43, 45, 50, 70, 100,
106
nucleotide sequence, 94
nucleus, 10, 14
nutrients, 2

O
obesity, 5
obstructive sleep apnea, 14
oligonucleotide arrays, 70, 82, 92
oral cavity, 23
organ(s), 2, 95
orofacial clefts, vii, 6, 18, 19, 20, 22, 25, 28,
32, 33, 35, 36, 38
oxygen, 2

P
paints, 62
pairing, 61
palatal clefts, ix, 18, 19, 21, 25, 26, 31, 33,
34
palate, ix, 17, 18, 19, 20, 21, 22, 23, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39
parallel, 44, 50, 73, 95, 106
parental consent, 88
parents, x, 18, 34, 41, 44, 54, 64, 70, 72, 80,
81, 82, 85, 86, 88, 89, 96
pathogenesis, 102
pathophysiology, 11
pathways, ix, 8, 9, 10, 15
PCR, 43, 44, 48, 55, 58, 68, 73, 77, 82, 90,
91, 94, 96, 99
pedigree, 86, 88
penetrance, 72, 80
perinatal, 3, 18
peripheral blood, 45, 47, 106
permit, 45
phenotype(s), x, 18, 54, 55, 62, 63, 66, 72,
79, 80, 82, 85, 87, 93, 96, 98, 99, 103
pilot study, 30, 90
placenta, vii, viii, 2, 3, 5, 10, 42, 55, 58, 59,
61, 93, 95, 106
placenta previa, viii, 2
placental malformations, viii, 2
plasticity, 10
platform, x, xi, 54, 70, 72, 73, 74, 77, 90,
96, 100
point mutation, 49
polycyclic aromatic hydrocarbon, 10
polyhydramnios, viii, 2
polymerase chain reaction, 101
polymorphism(s), 43, 44, 72, 87
polyploidy, 71
population, viii, 1, 13, 19, 20, 21, 22, 23, 35,
36, 58, 62, 86
postnatal exposure, 12
postnatal management, vii, x, 18
Prader-Willi syndrome, 99
preeclampsia, 49, 50

113

Index
pregnancy, vii, viii, ix, x, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 13, 14, 15, 16, 34, 48, 49,
54, 55, 57, 58, 59, 60, 73, 80, 81, 82, 83,
85, 86, 88, 90, 95, 96, 104, 105
pregnant women, vii, viii, x, 1, 2, 3, 4, 5, 20,
32, 41, 42, 47, 55, 56, 74, 89, 94
premature delivery, vii, viii, 2
premature rupture of membranes, vii, viii, 2
prematurity, 3
preterm birth, viii, 2
preterm delivery, 3
prevention, 61
probe, 23, 68, 100
prognosis, 18, 57
public health, vii, 1

R
RB1, 81
receptors, 11, 13, 15
reciprocal translocation, 62, 93
recombination, 65
reconstruction, 25, 30
recurrence, 64, 70, 83, 85
registries, 19
replication, 10
reproductive age, viii, 1
resolution, xi, 54, 55, 66, 67, 70, 71, 73, 74,
77, 82, 85, 90, 96, 102
resources, 79, 82
reticular activating system, 10
ring chromosome, 67
risk(s), viii, x, 1, 2, 3, 4, 6, 12, 13, 18, 19,
20, 22, 31, 35, 41, 42, 43, 48, 49, 50, 54,
55, 56, 57, 58, 62, 64, 68, 70, 80, 81, 82,
83, 85, 93, 94, 96, 97, 103
risk assessment, 93
risk factors, 6, 12, 19
RNA, 50
Robertsonian translocation, 65, 93, 105
rubella, 57
Russia, 7

S
safety, x, 41
sensitivity, 4, 21, 22, 26, 32, 43, 44, 70, 94
sensitization, 6
sequencing, vii, x, 41, 42, 44, 45, 46, 47, 48,
50, 51, 94, 95, 106, 107
serotonin, 11
serum, 48, 50, 56, 57, 61, 106
sex, 43, 49, 60, 95
sex chromosome, 60, 95
showing, 33, 66, 84, 85, 90
signs, 9, 32
skeleton, 32
skin, 58, 103
sleep disturbance, vii, ix, 7, 8, 10, 12, 14
sleep schedule, ix, 8
sleep stage, 11
smoke exposure, 4, 6
smoking, viii, ix, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16
smoking cessation, 5, 13
SNP, 44, 45, 47, 65, 70, 74, 75, 77, 90, 91,
92, 93, 104, 105
snuff during pregnancy, viii, 1, 3, 4
social class, 5
society, 87
solid tumors, 100
solution, 68
somatic cell, 46
South Africa, 19
Spain, vii, viii, 1, 4, 5
speech, 103
sperm, 61
spina bifida, 104
spinal cord, 11, 13
spontaneous abortion, vii, viii, 2, 67, 89, 97,
99, 104, 105
state(s), 13, 77, 88, 93
statistics, 19
stillbirth, 57, 91
stress, 9, 13
STRs, 68
structural variation, 101
structure, 27

114

Index

substance abuse, 15
substitutes, 60
substrate, 71
success rate, 89, 92
sudden infant death syndrome (SIDS), ix, 6,
8, 11, 12, 13
survival, 11, 14
susceptibility, 78
Sweden, 36
symmetry, 25
symptoms, 67, 81
synapse, 11
syndrome, 12, 15, 20, 21, 61, 63, 65, 67, 80,
83, 84, 86, 87, 94, 95, 99, 102, 103, 104,
107

T
tachycardia, 61
Taiwan, 19, 35
talipes equinovarus, 58
tar, viii, 1
target, 70
techniques, ix, 18, 19, 22, 23, 26, 27, 31, 32,
34, 55, 56, 66, 90, 97
technology(s), vii, ix, x, 17, 34, 43, 48, 53,
69, 73, 94, 102
testing, x, xi, 48, 50, 54, 56, 57, 67, 69, 70,
73, 74, 77, 81, 82, 88, 90, 95, 96, 99, 106
thalassemia, 43, 49, 55, 57
tissue, xi, 25, 54, 60, 89
tobacco, vii, ix, 1, 5, 6, 8, 10, 12, 14, 15, 16
tobacco smoke, ix, 5, 6, 8, 10, 12
tobacco smoking, 15
toxic effect, 10
training, 23, 25
translocation, 62, 64, 81, 82, 83, 85, 91, 93,
95, 98, 103
transmission, 83
transport, 2
treatment, 19, 35
tremor, 9
trial, 48, 97
triggers, 11

trisomy, 21, 42, 44, 50, 51, 60, 74, 94, 95,
105, 106
Tyrosine, 83

U
ultrasonography, 37, 57
ultrasound, vii, ix, 5, 17, 18, 20, 21, 22, 23,
25, 28, 31, 32, 33, 35, 36, 37, 38, 39, 42,
55, 56, 57, 58, 59, 63, 67, 72, 73, 74, 77,
78, 80, 81, 82, 83, 86, 88, 90, 91, 96, 98,
101, 102, 104, 105
umbilical cord, 55, 59
United Kingdom (UK), 21, 36, 97
USA, 48, 50, 106
uterus, 58
uvula, 26, 30

V
variable expressivity, x, 54, 80, 96
variables, 82, 91
variations, 19, 23, 34, 67, 69, 86, 103, 105
victims, 12
villus, 55, 97, 98
vinyl chloride, 10
viral infection, 57
vision, 107
visualization, 19, 27, 30, 33, 34, 35, 37, 56,
83

W
withdrawal, 15
women smokers, viii, 2
World Health Organization (WHO), 19, 35
wound healing, 34
wrists, 83

Y
Y chromosome, 43