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OBSTETRICS AND
GYNECOLOGY ADVANCES
AARON HENDERSON
EDITOR
New York
Copyright © 2015 by Nova Science Publishers, Inc.
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Preface vii
Chapter 1 Maternal Smoking in Pregnancy and Its Impact
on Placental and Fetal Development 1
Emilio González-Jiménez
Chapter 2 Maternal Smoking during Pregnancy and the Risk
of Sleep Disturbances in Infants 7
Igor A. Kelmanson
Chapter 3 Prenatal Screening and Diagnosis of Facial Clefts:
Where Are We Now? 17
William W. K. To and Meliza C. W. Kong
Chapter 4 Noninvasive Prenatal Diagnosis by Sequencing
Circulating Fetal DNA 41
Liang Xu and Rui Shi
Chapter 5 Array CGH in Prenatal Diagnosis 53
Paola Evangelidou, Philippos C. Patsalis
and Carolina Sisman
Index 107
PREFACE
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.
Αrray 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
the resolution
the type of platform to be used
ethical issues including coincidental and of unclear significance
findings
Chapter 1
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 subcoriónica 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 … 3
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.
REFERENCES
[1] Aurrekoetxea JJ, Murcia M, Rebagliato M, Fernández-Somoano A,
Castilla AM, Guxens M, López MJ, Lertxundi A, Espada M, Tardón A1,
Ballester F, Santa-Marina L. Factors associated with second-hand smoke
exposure in non-smoking pregnant women in Spain: self-reported
exposure and urinary cotinine levels. Sci. Total Environ. 2014; 470-
471:1189-96.
[2] Jiménez-Muro Franco A. Estudio de tabaquismo en mujeres
embarazadas: eficacia de un programa de intervención proactivo y
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[3] Rogers JM. Tobacco and pregnancy. Reprod. Toxicol. 2009; 28(2):152-
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[4] Becoña E. Tabaco. Prevención y Tratamiento. Madrid: Pirámide, 2006.
[5] Jiménez-Muro A, Samper MP, Marqueta A, Rodríguez G, Nerín I.
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between Spanish and immigrant pregnant women. Gac. Sanit. 2012;
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Maternal Smoking in Pregnancy and Its Impact on Placental … 5
[18] Von Mandach U. Drug use in pregnancy. Ther. Umsch. 2005; 62(1): 29-
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[19] Nechanská B, Mravčík V, Sopko B, Velebil P. Pregnant women and
mothers using alcohol, tobacco and illegal drugs. Ceska Gynekol. 2012;
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[20] Scherer G, Conze C, Meyerinck L, Sorsa M, Adlkofer F. Importance of
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[21] Aguirre Camposano V. Smoking during pregnancy: Effects on
respiratory children‘s health. Rev. Chil. Enf. Respir. 2007; 23: 173-178
[22] Cnattingius S. The epidemiology of smoking during pregnancy: smoking
prevalence, maternal characteristics, and pregnancy outcomes. Nicotine
Tob. Res. 2004; 6: S125-S140.
[23] Difranza JR, Aligne CA, Weitzman M. Prenatal and postnatal
environmental tobacco smoke exposure and children's health. Pediatrics.
2004; 113: 1007-15.
[24] Sehested LT, Pedersen P. Prognosis and risk factors for intrauterine
growth retardation. Dan. Med. J. 2014; 61(4): A4826.
[25] Lieberman E, Torday J, Barbieri R, Cohen A, Van Vunakis H, Weiss
ST. Association of intrauterine cigarette smoke exposure with indices of
fetal lung maturation. Obstet. Gynecol. 1992; 79: 564-70.
[26] Hylkema MN, Blacquière MJ. Intrauterine effects of maternal smoking
on sensitization, asthma, and chronic obstructive pulmonary disease.
Proc. Am. Thorac. Soc. 2009; 6(8): 660-2.
[27] Elliot J, Vullermin P, Robinson P. Maternal cigarette smoking is
associated with increased inner airway wall thickness in children who
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1998; 158: 802-6.
[28] Sánchez Agudo L. El fumador pasivo. Adicciones. 2004; 16 (Supl 2):
83-99.
[29] Morán-Barroso VF. Efectos del tabaquismo materno en el desarrollo
prenatal. Bol. Med. Hosp. Infant Mex. 2007; 64: 69-71.
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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
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.
8 Igor A. Kelmanson
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).
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
REFERENCES
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Syndrome and prenatal maternal smoking: rising attributed risk in the
Back to Sleep era. BMC Medicine, 3(1), 4. doi: 10.1186/1741-7015-3-4.
Chen, W. J., Parnell, S. E., & West, J. R. (1998). Neonatal alcohol and
nicotine exposure limits brain growth and depletes cerebellar Purkinje
cells. Alcohol, 15(1), 33-41.
Maternal Smoking during Pregnancy and the Risk ... 13
Chernick, V., Childiaeva, R., & Ioffe, S. (1983). Effects of maternal alcohol
intake and smoking on neonatal electroencephalogram and anthropometric
measurements. Am J Obstet Gynecol, 146(1), 41-47.
Cohen, G., Han, Z. Y., Grailhe, R., Gallego, J., Gaultier, C., Changeux, J. P.
(2002). beta 2 nicotinic acetylcholine receptor subunit modulates
protective responses to stress: A receptor basis for sleep-disordered
breathing after nicotine exposure. Proc Natl Acad Sci U S A, 99(20),
13272-13277. doi: 10.1073/pnas.192463599
Cohen, G., Roux, J. C., Grailhe, R., Malcolm, G., Changeux, J. P., &
Lagercrantz, H. (2005). Perinatal exposure to nicotine causes deficits
associated with a loss of nicotinic receptor function. Proc Natl Acad Sci U
S A, 102(10), 3817-3821. doi: 10.1073/pnas.0409782102
Dani, J. A. (2001). Overview of nicotinic receptors and their roles in the
central nervous system. Biol Psychiatry, 49(3), 166-174.
Dempsey, D. A., & Benowitz, N. L. (2001). Risks and benefits of nicotine to
aid smoking cessation in pregnancy. Drug Saf, 24(4), 277-322.
DiFranza, J. R., Aligne, C. A., & Weitzman, M. (2004). Prenatal and Postnatal
Environmental Tobacco Smoke Exposure and Children's Health.
Pediatrics, 113(4), 1007-1015.
Ekblad, M., Korkeila, J., & Lehtonen, L. (2015). Smoking during pregnancy
affects foetal brain development. Acta Paediatrica, 104(1), 12-18. doi:
10.1111/apa.12791
Franco, P., Groswasser, J., Hassid, S., Lanquart, J. P., Scaillet, S., & Kahn, A.
(1999). Prenatal exposure to cigarette smoking is associated with a
decrease in arousal in infants. J Pediatr, 135(1), 34-38.
Frank, M. G., Srere, H., Ledezma, C., O'Hara, B., & Heller, H. C. (2001).
Prenatal nicotine alters vigilance states and AchR gene expression in the
neonatal rat: implications for SIDS. Am J Physiol Regul Integr Comp
Physiol, 280(4), R1134-1140.
Fried, P. A., Watkinson, B., Dillon, R. F., & Dulberg, C. S. (1987). Neonatal
neurological status in a low-risk population after prenatal exposure to
cigarettes, marijuana, and alcohol. J Dev Behav Pediatr, 8(6), 318-326.
Haggart, M., & Giblin, M. J. (1988). Passive smoking and colic-like behaviour
in babies. Health Visit, 61(3), 81-82.
Hellstrom-Lindahl, E., Gorbounova, O., Seiger, A., Mousavi, M., & Nordberg,
A. (1998). Regional distribution of nicotinic receptors during prenatal
development of human brain and spinal cord. Brain Res Dev Brain Res,
108(1-2), 147-160.
14 Igor A. Kelmanson
Chapter 3
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 defects—particularly those of the palate—three- 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.
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].
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 low-
risk 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].
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 operator‘s 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].
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 over-
diagnosed 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
in the coronal plane at the base of the retronasal triangle, and the secondary
palate by virtual navigation in the axial plane.
Figure 3. Lip cleft, alveolar cleft and palate cleft as visualized by 3D USG surface
rendered images.
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].
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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Obstet. Gynecol. 2001; 18:432-436.
[2] Tonni G, Centini G, Rosignoli L. Prenatal screening for fetal face and
clefting in a prospective study on low-risk population: can 3- and 4-
dimensional ultrasound enhance visualization and detection rate? Oral
Surg. Oral Med. Oral Pathol. Oral Radiol. Endo. 2005; 100:420-426.
[3] Bergé SJ, Plath H, Van de Vondel PT, Appel T, Niederhagen B, Von
Lindern JJ, Reich RH, Hansmann M. Facial cleft lip and palate:
sonographic diagnosis, chromosomal abnormalities, associated
anomalies and postnatal outcome in 70 fetuses. Ultrasound Obstet.
Gynecol. 2001; 18:422-431.
[4] Maarse W, Bergé SJ, Pistorius L, van Barneveld T, Kon M, Breugem C,
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ultrasound in detecting prenatal cleft lip and palate: a systematic
review. Ultrasound Obstet. Gynecol. 2010; 35:495-502.
[5] Heinrich A, Proff P, Michel T, Ruhland F, Kirbschus A, Gedrange T.
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and infant care. J. Craniomaxillofac. Surg. 2006; 34 Suppl 2:14S-16S.
[6] Oosterkamp BC, Dijkstra PU, Remmelink HJ, van Oort RP, Goorhuis-
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[7] World Health Organization. Global registry and database on craniafacial
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[9] Pavri S, Forrest CR. Demographics of orofacial clefts in Canada from
2002-2008. The Cleft. Palat. Craniofac. J. 2013; 50: 224-230.
36 William W. K. To and Meliza C. W. Kong
[20] Nyberg DA, Hegge FN, Kramer D, Mahony BS, Kropp RJ. Premaxillary
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Ultrasound Med. 1993; 12: 331-5.
[21] Bronshtein M, Blumenfeld I, Kohn J, Blumenfeld Z. Detection of cleft
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[22] Sherer DM, Abramowicz JS, Jaffe R, Woods JR Jr. Cleft palate:
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Diagn. 1993; 13: 953-956.
[23] Kurjak A, Azumendi G, Andonotopo W, Salihagic-Kadic A. Three- and
four-dimensional ultrasonography for the structural and functional
evaluation of the fetal face. Am. J. Obstet. Gynecol. 2007;196: 16-28.
[24] Ensing S, Kleinrouweler CE, Maas SM, Bilardo CM, van der Horst
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diagnosis and management of fetal facial clefts. Ultrasound Obstet.
Gynecol. 2014; 44: 154-159.
[25] Timor-Tritsch IE, Platt LD. Three-dimensional ultrasound experience in
obstetrics. Curr. Opin. Obstet. Gynecol. 2002;14: 569-75.
[26] Leung KY, Ngai CS, Tang MH. Facial cleft or shadowing artifact?
Ultrasound Obstet. Gynecol. 2006;27:231-272.
[27] Guenot C, Baud D, Lepigeon K, Francini K, Rossier MC, Hohlfeld P,
Vial Y. Inexperienced sonographers can successfully visualize and
assess a three-dimensional image of the fetal face using a standardized
ultrasound protocol. Fetal. Diagn. Ther. 2013; 34: 96-102.
[28] Wilhelm L, Borgers H. The ‗equals sign‘: a novel marker in the
diagnosis of fetal isolated cleft palate. Ultrasound Obstet. Gynecol.
2010;36: 439-44.
[29] Campbell S, Lees C, Moscoso G, Hall P. Ultrasound antenatal diagnosis
if cleft palate by a new technique: the 3D ―reverse face‖ view.
Ultrasound Obstet. Gynecol. 2005;25: 12-8.
[30] Platt LD, Devore GR, Pretorius DH. Improving cleft palate/ cleft lip
antenatal diagnosis by 3-dimensional sonography: the ―flipped face‖
view. J. Ultrasound Med. 2006; 25:1423-30.
[31] Pilu G, Segata M. A novel technique for visualization of the normal and
cleft fetal secondary palate: angled insonation and three-dimensional
ultrasound. Ultrasound Obstet. Gynecol. 2007;29: 166-9.
[32] Ten PM, Pedregosa JP, Santacruz B, Adiego B, Barron E, Sepulveda W.
Three-dimensional ultrasound diagnosis of cleft palate: ‗reverse face‘,
38 William W. K. To and Meliza C. W. Kong
Chapter 4
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
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.
qPCR
SRY gene, RHD gene, β-globin gene mutation congenital adrenal hyperplasia [15, 16, 35],
PCR + MS
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 448 severe recessive childhood diseases [30]
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).
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 father‘s
age contributes to the diversity in the mutation rate of SNPs; thus the father‘s
genetic information should be added to enhance the analysis of the fetal
genome.
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
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attention. Nat. Rev .Genet 2006;7:277-82.
[2] Costa T, Scriver CR, Childs B. The effect of Mendelian disease on
human health: a measurement. Am. J. Med. Genet 1985;21:231-42.
[3] Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B.
Randomised controlled trial of genetic amniocentesis in 4606 low-risk
women. Lancet 1986;1:1287-93.
[4] ACOG Practice Bulletin No. 88, December 2007. Invasive prenatal
testing for aneuploidy. Obstet. Gynecol 2007;110:1459-67.
[5] Jauniaux E, Pahal GS, Rodeck CH. What invasive procedure to use in
early pregnancy. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol
2000;14:651-62.
[6] Chan KC, Zhang J, Hui AB, et al. Size distributions of maternal and
fetal DNA in maternal plasma. Clin. Chem 2004;50:88-92.
[7] Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in
maternal plasma and serum. Lancet 1997;350:485-7.
[8] Guibert J, Benachi A, Grebille AG, Ernault P, Zorn JR, Costa JM.
Kinetics of SRY gene appearance in maternal serum: detection by real
time PCR in early pregnancy after assisted reproductive technique. Hum.
Reprod 2003;18:1733-6.
[9] Chim SS, Tong YK, Chiu RW, et al. Detection of the placental
epigenetic signature of the maspin gene in maternal plasma. Proc. Natl.
Acad. Sci. USA 2005;102:14753-8.
[10] Masuzaki H, Miura K, Yoshiura KI, Yoshimura S, Niikawa N, Ishimaru
T. Detection of cell free placental DNA in maternal plasma: direct
evidence from three cases of confined placental mosaicism. J. Med.
Genet 2004;41:289-92.
Non-Invasive Prenatal Diagnosis ... 49
[11] Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid
clearance of fetal DNA from maternal plasma. Am. J. Hum. Genet
1999;64:218-24.
[12] Rijnders RJ, Christiaens GC, Soussan AA, der Schoot CE v. Cell-free
fetal DNA is not present in plasma of nonpregnant mothers. Clin. Chem.
2004;50:679-81; author reply 681.
[13] Smid M, Galbiati S, Vassallo A, et al. No evidence of fetal DNA
persistence in maternal plasma after pregnancy. Hum. Genet
2003;112:617-8.
[14] Nygren AO, Dean J, Jensen TJ, et al. Quantification of fetal DNA by use
of methylation-based DNA discrimination. Clin. Chem 2010;56:1627-
35.
[15] Rijnders RJ, der Schoot CE v, Bossers B, de Vroede MA, Christiaens
GC. Fetal sex determination from maternal plasma in pregnancies at risk
for congenital adrenal hyperplasia. Obstet. Gynecol 2001;98:374-8.
[16] Chiu RW, Lau TK, Cheung PT, Gong ZQ, Leung TN, Lo YM.
Noninvasive prenatal exclusion of congenital adrenal hyperplasia by
maternal plasma analysis: a feasibility study. Clin. Chem. 2002;48:778-
80.
[17] Devaney SA, Palomaki GE, Scott JA, Bianchi DW. Noninvasive fetal
sex determination using cell-free fetal DNA: a systematic review and
meta-analysis. JAMA 2011;306:627-36.
[18] Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal D
status from maternal plasma: introduction of a new noninvasive fetal
RHD genotyping service. Transfusion (Paris) 2002;42:1079-85.
[19] Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal
diagnosis of myotonic dystrophy using fetal DNA obtained from
maternal plasma. Clin. Chem. 2000;46:301-2.
[20] Chiu RW, Lau TK, Leung TN, Chow KC, Chui DH, Lo YM. Prenatal
exclusion of beta thalassaemia major by examination of maternal
plasma. Lancet 2002;360:998-1000.
[21] Li Y, Di NE, Vitucci A, Zimmermann B, Holzgreve W, Hahn S.
Detection of paternally inherited fetal point mutations for beta-
thalassemia using size-fractionated cell-free DNA in maternal plasma.
JAMA 2005;293:843-9.
[22] Levine RJ, Qian C, Leshane ES, et al. Two-stage elevation of cell-free
fetal DNA in maternal sera before onset of preeclampsia. Am. J. Obstet
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50 Liang Xu and Rui Shi
[23] Lo YM, Leung TN, Tein MS, et al. Quantitative abnormalities of fetal
DNA in maternal serum in preeclampsia. Clin. Chem. 1999;45:184-8.
[24] Ng EK, Leung TN, Tsui NB, et al. The concentration of circulating
corticotropin-releasing hormone mRNA in maternal plasma is increased
in preeclampsia. Clin. Chem. 2003;49:727-31.
[25] Oudejans CB, Go AT, Visser A, et al. Detection of chromosome 21-
encoded mRNA of placental origin in maternal plasma. Clin. Chem.
2003;49:1445-9.
[26] Ding C, Chiu RW, Lau TK, et al. MS analysis of single-nucleotide
differences in circulating nucleic acids: Application to noninvasive
prenatal diagnosis. Proc Natl Acad Sci U S A 2004;101:10762-7.
[27] Lo YM, Tsui NB, Chiu RW, et al. Plasma placental RNA allelic ratio
permits noninvasive prenatal chromosomal aneuploidy detection. Nat
Med. 2007;13:218-23.
[28] Chiu RW, Chan KC, Gao Y, et al. Noninvasive prenatal diagnosis of
fetal chromosomal aneuploidy by massively parallel genomic
sequencing of DNA in maternal plasma. Proc. Natl. Acad. Sci. USA
2008;105:20458-63.
[29] Chen EZ, Chiu RW, Sun H, et al. Noninvasive prenatal diagnosis of fetal
trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PLOS
ONE 2011;6:e21791.
[30] Bell CJ, Dinwiddie DL, Miller NA, et al. Carrier testing for severe
childhood recessive diseases by next-generation sequencing. Sci. Transl
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invasive prenatal measurement of the fetal genome. Nature
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[32] Kitzman JO, Snyder MW, Ventura M, et al. Noninvasive whole-genome
sequencing of a human fetus. Sci. Transl. Med. 2012;4:137ra76.
[33] Kong A, Frigge ML, Masson G, et al. Rate of de novo mutations and the
importance of father's age to disease risk. Nature 2012;488:471-5.
[34] Goldstein DB. Growth of genome screening needs debate. Nature
2011;476:27-8.
[35] Costa JM, Benachi A, Gautier E: New strategy for prenatal diagnosis of
X-linked disorders. N. Engl. J. Med. 2002; 346: 1502.
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status by molecular analysis of maternal plasma. N. Engl. J. Med. 1998;
339: 1734-1738.
Non-Invasive Prenatal Diagnosis ... 51
Chapter 5
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.
Αrray 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
54 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
the resolution
the type of platform to be used
ethical issues including coincidental and of unclear significance
findings
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
56 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
In the first trimester maternal serum screening can check levels of free β-
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.
Therefore clinicians and patients should weigh the relative risks and
benefits of invasive prenatal diagnosis performed later as compared to earlier
in pregnancy.
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 procedure-
induced 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
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.
βHCG PAPP- A
Abnormality Sonographic markers
(2MoM) (0,5 MoM)
Trisomy 21 Increased Decreased Increased NT, absent nasal bone in
60-70% of trisomies
Trisomy 18 Decreased Decreased Early onset IUGR, relative
bradycardia, associated exomphalos in
30% of the cases
Trisomy 13 Decreased Decreased Fetal tachycardia in 60% of cases,
early onset IUGR, holoprocencephaly
or exomphalos in 30% of cases
Turner Normal Lower Fetal tachycardia in 50% of cases,
syndrome early onset IUGR
Triploidy- Greatly Mildly Early onset asymmetrical IUGR,
diandric increased decreased relative bradycardia,
holoprocencephaly, exomphalos or
posterior fossa cyst in 40% of cases,
molar changes in the placenta in 30 %
of cases
Triploidy- Markedly Markedly Early onset asymmetrical IUGR,
digynic decreased decreased relative bradycardia,
holoprocencephaly, exomphalos or
posterior fossa cyst in 40% of cases,,
molar changes in the placenta in 30 %
of cases
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
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.
7.2.2.1. 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, Miller-
Dieker 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
64 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
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
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
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 stand-
alone 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
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].
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].
Array CGH in Prenatal Diagnosis 75
Table 9.2.1. Comparison between various studies which used array CGH
in Prenatal diagnosis
Karyotype/
Clinical Significance
Study Array Type Reason for Results
of Results
Referral
Kleeman Signature Normal 4/50 abnormal 2% clinically
et al., 2009 prenatal targeted karyotype, significant, 6%
[63] BAC chip V, sonographic inherited or benign
signature whole anomalies variant
genome chip
Vialard Targeted Normal 4/37 abnormal 10.8% clinically
et al., 2009 Genosensor karyotype, significant
[59] BAC/PAC array multiple
congenital
abnormalities
Bi et al., 2008 BCM V6 Normal 3/15 abnormal 13% clinically
[57] oligonucleotide karyotype, significant, 7%
array maternal age, inherited or benign
sonographic variant
anomalies,
family history,
miscarriages
Shaffer Prenatal targeted 149/151 normal 15/151 1.3% clinically
et al., 2008 BAC array karyotype, abnormal significant, 8%
[60] maternal age, benign, 0.5% unclear
sonographic significance
anomalies,
family history,
parental anxiety
Sahoo et al., BCM V4 targeted 93/98 normal 5/98 abnormal 5% clinically
2006 [56] BAC array karyotype, of which one significant
maternal age, had additional
sonographic abnormalities
anomalies,
family history
Tyreman GeneChip SNP sonographic 35/106 9% likely pathogenic,
et al., 2009 whole genome abnormalities abnormal 12% likely benign,
[62] oligonucleotide 13% unclear
array significance
Coppinger Signature V 4.0, Normal Whole Whole genome: 2.7%
et al., 2009 prenatal targeted karyotype, genome: clinically significant,
[58] BAC array and maternal age, 22/180 0.5% unclear
whole genome sonographic abnormal. significance, 8.8%
array anomalies, Targeted: 7/62 benign variants.
family history, abnormal Targeted: 0.9%
anxiety clinically significant,
0.5 unclear
significance, 8%
benign variants
76 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
Karyotype/
Clinical Significance
Study Array Type Reason for Results
of Results
Referral
Fiorentino Cytochip Focus Maternal age, 34/1037 3.3% clinically
et al., 2011 BAC array sonographic abnormal significant, 13%
[65] anomalies, benign variants.
family history,
anxiety
Scott et al. Targeted version Maternal age, 156/1049 11.7%
2013 [51] of 8x60K sonographic abnormal Numerical/Structural
oligonucleotide anomalies, abnormalities,
Agilent array family history, 3.1% CNVs
anxiety undetectable by G-
Banding of which:
1.2% pathogenic,
0.2% VOUS,0.9%
benign, 0.57% carrier
status of disease
Evangelidou Oligonucleotide 60/64 Normal 17/64 11% Clinically
et al., arrays 105K or and sonographic Abnormal significant changes
2013[66] 180K anomalies, 4/17 15.6% VOUS
Balanced Confirmation determined to be
karyotype with of previous familial CNVs
or without abnormal unrelated to the RFR
sonographic results in 100%
anomalies,
4/64 Abnormal
karyotype or
MLPA
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].
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
80 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
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), Dolichyl-
Phosphate 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
84 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
expected, chromosomal analysis could not detect the deletion as it was 2.6 Mb
in size and beyond this assay‘s 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.
Array CGH in Prenatal Diagnosis 85
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.
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 non-
targeted 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.
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.
92 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
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].
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,
96 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
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102 Paola Evangelidou, Philippos C. Patsalis and Carolina Sismani
amplitude, 11
# anatomy, 19, 27, 30
aneuploid, 94
3D images, 25
aneuploidy, 28, 31, 44, 48, 50, 57, 67, 68,
89, 90, 97, 99, 104, 106
A antigen, 43
anxiety, 70, 74, 75, 76, 80, 86
abuse, 48 apnea, 11
acetylcholine, 11, 13 apoptosis, 42
acid, viii, 2, 70 arousal, ix, 8, 11, 13, 14
acrocentric chromosome, 62 arteries, 3
addictive, viii, 1 arterioles, 3
adrenal hyperplasia, 43 Asian countries, 19
adults, viii, 7, 8 assessment, 23, 25, 26, 33, 34, 35, 36, 38,
advancement(s), vii, x, 41 51, 72
adverse effects, vii, viii, 2, 9 asthma, 6
aesthetic, 18 asymptomatic, 72
African American women, 15 audit, 30
age, 9, 22, 23, 25, 47, 50, 56, 74, 75, 76, 95, autism, x, 53
98, 106 awareness, 12, 93
agonist, 11
alcohol consumption, 15
B
allele, 43, 45, 79
alters, 3, 13
babbling, 9, 15
alveolar ridge, 22, 23, 25, 26, 27, 29, 36
BAC, 69, 70, 71, 73, 74, 75, 76, 81, 89, 90,
alveoli, 3
92
alveolus, 18, 21, 22, 25, 36
base, 29, 43, 44
ammonia, 10
base pair, 44
amniocentesis, x, 41, 48, 58, 59
basement membrane, 2
amnion, 58
behavioral problems, 103
amniotic fluid, viii, 2, 55, 58, 66, 93
108 Index
Belgium, 95, 107 children, x, 6, 14, 15, 16, 18, 21, 34, 53, 64,
benefits, 13, 57, 91 98, 100
benign, 65, 72, 73, 74, 75, 76, 77, 78, 79, China, 41
80, 81, 82, 87, 89, 98, 103 chorion, 97
biochemistry, 57, 61 chorionic villi, 58, 60
biological processes, 59 chorionic villus sampling, x, 41, 42, 86
biosynthesis, 11, 12 chromosomal abnormalities, 35, 55, 68, 79,
birth rate, 20 90, 101, 104
birth weight, 3, 20 chromosome, 43, 44, 46, 50, 58, 60, 61, 62,
births, 18, 19, 22, 65, 67 63, 65, 67, 68, 69, 70, 74, 80, 81, 82, 83,
bleeding, vii, viii, 2 84, 85, 87, 93, 94, 97, 98, 99, 100, 101,
blood, viii, 1, 2, 43, 45, 48, 51, 55, 59, 73, 103, 104, 105
95, 106 chromosome 10, 74
blood flow, viii, 2, 3 chronic obstructive pulmonary disease, 6
blood group, 43 cigarette smoke, viii, 6, 7, 8
blood pressure, viii, 2 cigarette smoking, vii, viii, 6, 7, 8, 12, 13,
bloodstream, 2 14, 15, 16
bone(s), 32, 34, 56, 61, 81 circulation, 2, 10, 73
bone growth, 34 classification, x, 22, 33, 54, 72, 80, 96
bradycardia, 61 cleft lip, ix, 3, 17, 19, 20, 21, 22, 23, 25, 26,
brain, viii, 1, 10, 11, 12, 13, 15, 16, 32, 38, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38,
100 39
brain abnormalities, 100 cleft palate, ix, 3, 17, 19, 20, 22, 23, 25, 26,
brain growth, 12 28, 31, 32, 36, 37, 38, 104
brain structure, 10 clinical diagnosis, 100
Brazil, 35 clinical disorders, 67
breathing, ix, 7, 11, 13, 14 clone, 90
cognitive development, vii, viii, 7, 8
colic, 9, 13
C Comparative Genomic Hybridization
(CGH), v, vii, x, xi, 53, 54, 55, 58, 62,
caesarean section, 95
64, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
calcification, 2
77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 89,
cancer, 69, 81, 89, 103
90, 91, 92, 93, 95, 96, 97, 98, 100, 101,
carbon monoxide, viii, 1, 10
102, 103, 104, 105
catecholamines, viii, 2, 3
complement, 18, 38, 55, 65, 82
cDNA, 71
complexity, 98
cell line, 65, 105
complications, 3, 5, 42, 57, 59
central nervous system (CNS), viii, 2, 13,
conception, 59, 60
15, 32, 33, 73
concordance, 91
cervix, 58, 73
congenital adrenal hyperplasia, 49
challenges, xi, 12, 54, 72, 80, 101, 102
congenital anomalies, x, 18, 53, 62, 81, 100
chemicals, viii, 1, 10
congenital malformations, 19
child development, 14
congenital structural abnormalities, ix, 17
childhood, 44, 50, 55
consent, 79, 104
Index 109
construction, 46 DNA, v, 10, 14, 41, 42, 43, 44, 45, 46, 47,
consumption, vii, viii, 1, 2, 3, 4 48, 49, 50, 51, 58, 59, 60, 62, 67, 68, 69,
contamination, 60, 68 70, 71, 73, 86, 92, 94, 95, 96, 101, 105,
controversies, 105 106
correlation, x, 54, 66, 79, 96 DNA sequencing, 50, 51
corticotropin, 50 DNA testing, 106
cost, 25, 48, 94 dopamine, 11
cotinine, 4, 14 dopaminergic, 11
counseling, 25, 70, 72, 81, 86, 88 dosage, xi, 54, 62, 69, 97
covering, 69 Down syndrome, 42, 94, 95, 106
culture, 60, 82, 90, 91, 97 drugs, 6
cyst, 61 dysmorphism, x, 53
cystic fibrosis, 55, 57
cytogenetics, xi, 54, 62, 63, 64, 67, 69, 70,
71, 73, 74, 77, 90, 92, 95, 96, 106 E
cytomegalovirus, 57
electroencephalogram, 8, 13
emergency, 95
D endocrine, 3
environmental tobacco, 6
database, xi, 35, 54, 79, 83, 86, 97 epidemic, vii, 1
deaths, 42 epidemiology, 6, 36
defects, ix, 3, 17, 18, 19, 21, 27, 56, 58, 71, epigenetic modification, 16
103 epigenetics, 14
deficiency, viii, 2, 103 epiglottis, 26
deficit, viii, 2 epinephrine, 11
deoxyribonucleic acid, 10 ethical issues, xi, 54
deposition, 2 ethics, 104
detectable, 77 euploid, 44, 94, 98
detection, ix, xi, 17, 18, 20, 21, 23, 25, 26, Europe, 19, 89
31, 32, 33, 34, 35, 36, 43, 44, 45, 48, 50, evoked potential, 9, 11, 14, 15
51, 54, 55, 66, 68, 70, 73, 75, 87, 90, 91, examinations, 20, 23
93, 94, 96, 99, 100, 101, 102, 103, 104, excitation, 9
105 exposure, ix, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14,
developed countries, vii, 1 15
diploid, 82 expressivity, 80
disability, x, 53, 55, 62, 64, 67, 103
discordance, 95
discrimination, 49, 82 F
disease gene, 83, 84
false negative, 95
diseases, x, 41, 42, 43, 44, 50, 78, 88, 89
false positive, 22, 23, 25, 29, 31, 95, 106
disorder, 43, 55, 57
families, 57
disposition, ix, 8, 10
family history, 21, 31, 74, 75, 76
dissociation, 58
family members, 83
distribution, 13, 20, 98
fetal abnormalities, 33
diversity, 22, 47
110 Index
morphology, 25, 32
mortality, vii, 1, 3, 18
P
mosaic, 60, 74, 78, 80, 93
paints, 62
mRNA, 43, 44, 50
pairing, 61
mucus, 101
palatal clefts, ix, 18, 19, 21, 25, 26, 31, 33,
mutant, 43
34
mutation(s), x, 41, 43, 47, 50, 71, 98, 104,
palate, ix, 17, 18, 19, 20, 21, 22, 23, 25, 26,
107
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
mutation rate, 47, 98
38, 39
parallel, 44, 50, 73, 95, 106
N parental consent, 88
parents, x, 18, 34, 41, 44, 54, 64, 70, 72, 80,
necrosis, 42 81, 82, 85, 86, 88, 89, 96
nervous system, 9 pathogenesis, 102
Netherlands, 20, 25, 95, 107 pathophysiology, 11
neurological abnormalities, x, 53 pathways, ix, 8, 9, 10, 15
neurons, 10 PCR, 43, 44, 48, 55, 58, 68, 73, 77, 82, 90,
neuropathy, 81 91, 94, 96, 99
neurotransmission, 11 pedigree, 86, 88
neurotransmitter(s), ix, 8, 10, 11 penetrance, 72, 80
newborns, vii, ix, 7, 8, 20, 65 perinatal, 3, 18
nicotine, viii, 1, 2, 3, 7, 8, 10, 12, 13, 15, 16 peripheral blood, 45, 47, 106
nitrogen, 10 permit, 45
nonsmokers, viii, 2 phenotype(s), x, 18, 54, 55, 62, 63, 66, 72,
Norway, 20, 36 79, 80, 82, 85, 87, 93, 96, 98, 99, 103
nuclei, 67 pilot study, 30, 90
nucleic acid, vii, x, 41, 43, 45, 50, 70, 100, placenta, vii, viii, 2, 3, 5, 10, 42, 55, 58, 59,
106 61, 93, 95, 106
nucleotide sequence, 94 placenta previa, viii, 2
nucleus, 10, 14 placental malformations, viii, 2
nutrients, 2 plasticity, 10
platform, x, xi, 54, 70, 72, 73, 74, 77, 90,
96, 100
O point mutation, 49
polycyclic aromatic hydrocarbon, 10
obesity, 5 polyhydramnios, viii, 2
obstructive sleep apnea, 14 polymerase chain reaction, 101
oligonucleotide arrays, 70, 82, 92 polymorphism(s), 43, 44, 72, 87
oral cavity, 23 polyploidy, 71
organ(s), 2, 95 population, viii, 1, 13, 19, 20, 21, 22, 23, 35,
orofacial clefts, vii, 6, 18, 19, 20, 22, 25, 28, 36, 58, 62, 86
32, 33, 35, 36, 38 postnatal exposure, 12
oxygen, 2 postnatal management, vii, x, 18
Prader-Willi syndrome, 99
preeclampsia, 49, 50
Index 113
substance abuse, 15 trisomy, 21, 42, 44, 50, 51, 60, 74, 94, 95,
substitutes, 60 105, 106
substrate, 71 Tyrosine, 83
success rate, 89, 92
sudden infant death syndrome (SIDS), ix, 6,
8, 11, 12, 13 U
survival, 11, 14
ultrasonography, 37, 57
susceptibility, 78
ultrasound, vii, ix, 5, 17, 18, 20, 21, 22, 23,
Sweden, 36
25, 28, 31, 32, 33, 35, 36, 37, 38, 39, 42,
symmetry, 25
55, 56, 57, 58, 59, 63, 67, 72, 73, 74, 77,
symptoms, 67, 81
78, 80, 81, 82, 83, 86, 88, 90, 91, 96, 98,
synapse, 11
101, 102, 104, 105
syndrome, 12, 15, 20, 21, 61, 63, 65, 67, 80,
umbilical cord, 55, 59
83, 84, 86, 87, 94, 95, 99, 102, 103, 104,
United Kingdom (UK), 21, 36, 97
107
USA, 48, 50, 106
uterus, 58
T uvula, 26, 30
tachycardia, 61
Taiwan, 19, 35 V
talipes equinovarus, 58
variable expressivity, x, 54, 80, 96
tar, viii, 1
variables, 82, 91
target, 70
variations, 19, 23, 34, 67, 69, 86, 103, 105
techniques, ix, 18, 19, 22, 23, 26, 27, 31, 32,
victims, 12
34, 55, 56, 66, 90, 97
villus, 55, 97, 98
technology(s), vii, ix, x, 17, 34, 43, 48, 53,
vinyl chloride, 10
69, 73, 94, 102
viral infection, 57
testing, x, xi, 48, 50, 54, 56, 57, 67, 69, 70,
vision, 107
73, 74, 77, 81, 82, 88, 90, 95, 96, 99, 106
visualization, 19, 27, 30, 33, 34, 35, 37, 56,
thalassemia, 43, 49, 55, 57
83
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 W
tobacco smoking, 15
toxic effect, 10 withdrawal, 15
training, 23, 25 women smokers, viii, 2
translocation, 62, 64, 81, 82, 83, 85, 91, 93, World Health Organization (WHO), 19, 35
95, 98, 103 wound healing, 34
transmission, 83 wrists, 83
transport, 2
treatment, 19, 35
tremor, 9 Y
trial, 48, 97
triggers, 11 Y chromosome, 43