Genetics and Molecular Biology, 37, 1 (suppl), 194-209 (2014)

Copyright © 2014, Sociedade Brasileira de Genética. Printed in Brazil

www.sbg.org.br

Review Article

Human molecular cytogenetics: From cells to nucleotides

Mariluce Riegel1,2
1
Serviço de Genética Médica, Hospital de Clínicas, Porto Alegre, RS, Brazil.
2
Programa de Pós-Graduação em Genética e Biologia Molecular,
Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

Abstract
The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnor-
malities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an
intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH), a technol-
ogy linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the di-
mension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and
research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology
techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of
microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods,
cytogeneticists have used a range of technologies to study the association between visible chromosome rearrange-
ments and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable num-
ber of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful
and informative complement to other molecular and genomic approaches. This manuscript does not present a de-
tailed history of the development of molecular cytogenetics; however, references to historical reviews and experi-
ments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the
technologies used to identify chromosomal rearrangements and copy number changes, and the applications for
cytogenetics in biomedical diagnosis and research are presented and discussed.
Keywords: molecular cytogenetics, FISH, array-CGH, copy number variation, genomic disorders.

Introduction Steele and Breg Jr (1966) succeeded in culturing amniotic

Arnold (1879), Flemming (1882) and Hansemann fluid cells and karyotyping fetal chromosomes. In the
(1890) reported the first microscopic observations of hu- 1970s, an in vitro culture technique for chorionic villi was
man mitotic chromosomes in the late 1800s. However, de- developed (Hahnemann, 1974), and Niazi et al. (1981) and
cades passed before the precise modal chromosome num- Brambati and Simoni (1983) improved this culture tech-
ber in humans was determined. Until Eagle developed nique several years later. Cytogenetics in hematology and
specific culture media in 1955, the cytogenetic analysis of oncology initially used peripheral blood as a specimen due
chromosomes depended on spontaneously dividing cells. to technical difficulties in processing and culturing solid tu-
Tjio and Levan (1956), using cultured embryonic cells, mor tissue. Because the development of newer techniques
were the first researchers to report the correct number of and more adequate methods has continued to increase the
human chromosomes as 46. Moorhead et al. (1960) estab- resolution of chromosomes, human cytogenetics has
lished an in vitro culture method for the accumulation of di- evolved from a more basic science into a valuable strategy
viding cells using colchicine to arrest cells at metaphase. In for diagnosing prenatal, postnatal and acquired chromo-
the same year, Nowell (1960) discovered the mitogenic somal abnormalities. The introduction and successful ap-
property of phytohemagglutinin, resulting in further techni- plication of a variety of chromosome-staining techniques in
cal improvements, particularly the use of peripheral blood previous years and molecular cytogenetic methods in re-
cells. Both events significantly increased the number of cent years has tremendously improved the number of chro-
metaphase spreads available for chromosome analysis. mosomal abnormalities described. Since the first observa-
tion of an extra copy of chromosome 21 (Lejeune et al.,
Send correspondence to Mariluce Riegel. Serviço de Genética
Médica, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos
1959) in patients with Down syndrome, many more chro-
2350, 90035-003 Porto Alegre, RS, Brazil. E-mail: mosomal abnormalities, such as other trisomies, translo-
mriegel@hcpa.ufrgs.br. cations, inversions, insertions, deletions, duplications and
Riegel 195

complex chromosome rearrangements, have been de- chromosome anomaly (Philadelphia chromosome) was de-
scribed. Novel methods for investigating the mechanisms scribed in patients with chronic myeloid leukemia (Nowell,
underlying copy number changes, characterizing gene in- 1960). Subsequent technical improvements in cytogenetics
teractions and analyzing genes within copy number varia- included the use of phytohemagglutinin (a substance that
tions (CNVs) are now being explored. Because the majority stimulates the division of T lymphocytes in vitro) and the
of techniques have been developed to study human introduction of banding techniques at the end of the 1960s.
genomes, man has been by far the most extensively studied Banding techniques use chemical treatments to produce
organism in cytogenetics. An overview of the first years of differentially stained regions on chromosomes. The band-
human cytogenetics and descriptions of classical and mo- ing pattern is highly characteristic for each chromosome
lecular cytogenetic techniques applied to the study of chro- and facilitates the complete identification of the human
mosomal abnormalities and evaluate copy number changes karyotype.
are discussed in more detail below.
Chromosome Banding Techniques
The Beginning of Human Cytogenetics
With the possibility of more specific identification
Human cytogenetics research began in 1879 with the and detailed analyses of human chromosomes, a new phase
observations of the German pathologist Arnold, who exam- in cytogenetics began. The first method for the visualiza-
ined carcinoma and sarcoma cells because the voluminous tion of a pattern of bands on human chromosomes was
nuclei of these cells facilitated analysis. Later, Flemming Q-banding (Caspersson et al., 1968). Subsequently, G-ban-
and Hansemann were the first to examine human mitotic ding (Seabright 1971), a technique based on the application
chromosomes. In the late 19th century Waldeyer (1888) of trypsin (a proteolytic enzyme) using Giemsa staining,
proposed the word “chromosome”, which means, “colored was developed, and this method is still the most widespread
body” (from the Greek chroma = color and soma = body). cytogenetic method routinely used in clinical settings.
The use of colchicine for chromosome preparations was Classical cytogenetics became a traditional powerful diag-
first implemented in plant cytogenetics in the 1930s nostic tool for detecting genomic aberrations, including
(Blakeslee and Avery, 1937; Levan, 1938). This substance both gains and losses of segments of the genome and rear-
acts as a poison that inhibits spindle formation during mito- rangements within and between chromosomes. However,
sis, increasing the number of metaphase spreads available the resolution of standard cytogenetics techniques re-
for analysis in a preparation. The treatment of cells with a mained limited, with a count of approximately 400-500
hypotonic solution facilitated better chromosome spread- bands per haploid genome (Figure 1). The approaches de-
ing, leading to better definition for counting the chromo- scribed above facilitated the identification of structural
somes. Previous studies have shown that unspread and chromosomal aberrations of at least 5-10 Mb in size. The
tangled chromosomes make it difficult to count the number average resolution depends on different elements, such as
of mammalian chromosomes in a preparation (Matthey, the optical characteristics of the microscope, the complex
1949). An improved hypotonic treatment technique (hypo- manner in which the DNA is packaged into chromosomes
tonic shock) was then applied to examine lung fibroblasts and the quality of the metaphase preparations. The resolu-
in human embryos, thereby establishing the correct modal tion of the standard karyotype was improved after the intro-
number of 46 chromosomes in human diploid cells (Tjio duction of high-resolution banding based on the use of
and Levan, 1956). In decades prior to this discovery, a hu- synchronized lymphocyte cultures (Yunis, 1976). Using
man chromosome number of 48 had been described in a this technique, it was possible to increase the number of
number of reports (see Gartler, 2006). This number was cells in the pro-metaphase or prophase stages. Detailed
based on an examination of chromosome preparations of principles, protocols and potential applications for these
human spermatogonia, which suggested that humans had cytogenetic banding techniques have been summarized
48 chromosomes (Painter, 1923). elsewhere (Wegner, 1999).
Although only a few chromosome details were
known during the pre-banding era, the chromosomes them- Fluorescence in situ Hybridization (FISH) and
selves could be arranged in different groups based on their
Multiple Advances
sizes and centromere positions. Following the determina-
tion of the correct modal chromosome number, the identifi- The considerable gap between the limited resolution
cation of the first inherited chromosomal abnormality for observing chromosome structure through banding tech-
(aneuploidy) leading to human diseases in man was identi- niques (> 5 Mb, depending on the banding resolution ap-
fied. Lejeune et al. (1959) reported trisomy 21 in Down plied) at the light microscopy and gene levels was bridged
syndrome patients. Subsequently, the chromosomal abnor- after the introduction and application of several molecular
malities causing Klinefelter (47, XXY) and Turner (45, X) cytogenetic approaches. The first applications of molecular
syndromes were identified (Ford et al., 1959; Jacobs and techniques to chromosome slide preparations, called in situ
Strong, 1959). During the same period, the first acquired hybridization (ISH), were attempts to identify and locate
196 Human molecular cytogenetics

Figure 1 - Human Karyotype. GTG-banded male patient with a normal metaphase spread with approximately 550 bands.

specific nucleic acid sequences inside cells or on chromo- levels, making this technique applicable for both clinical
somes (Gall and Pardue, 1969; John et al., 1969). The ISH diagnosis and research. FISH has been a driving force in the
technique was based on the discovery that radioactively la- further development of cytogenetic techniques. The basic
beled ribosomal RNA hybridized to acrocentric chromo- principle of FISH is that a target DNA in cells, nuclei or
somes. The hybridization was visualized using autoradio- metaphase chromosomes is fixed and denatured on the sur-
graphy, which had been applied to human chromosomes face of the slide. The probe DNA must be labeled with a nu-
since the early 1960s (German and Bearn, 1961). The use of cleotide that is either conjugated to fluorescein (direct
ISH technology provided another dimension to the study of labeling) and/or a non-fluorescent hapten (indirect label-
chromosomes, facilitating the visualization of DNA or ing), and the probe is first denatured and pre-hybridized
complementary RNA sequences on chromosomes and in with unlabeled repetitive DNA. Before hybridization, the
cells at the molecular level. However, the use of this metaphase chromosome suspension and/or interphase nu-
method was limited due to the use of radioactive isotopes, clei are enzymatically pretreated to enhance accessibility to
highly repetitive DNA sequences and corresponding RNA the probe and reduce the amount of cytoplasm. The pre-
in the satellite regions of chromosomes and centromeres treated slide containing the target and probe DNA is heated
(Pardue and Gall, 1970). to denature the DNA. The prepared probe is subsequently
Subsequently, Langer et al. (1981) improved ISH applied to the slide for ~16-48 h at 37°C for hybridization.
with the development of a technique involving the use of a The speed of the hybridization between the probe and the
nonradioactive probe (such as biotin) for indirect labeling target DNA varies depending on the probe used. Post-
through nick translation. The hybridization (DNA probe hybridization washes remove unbound single-strand DNA
and target sequence) could be visualized through avidin or and non-specifically bound DNA from the slide. When a
streptavidin fluorescent labeling. The development of fluo- non-fluorescent hapten is used (e.g., biotin or digoxigenin),
rescent molecules led to direct (combined with a fluoro- the detection occurs through a fluorescence-coupled anti-
chrome) or indirect (through an intermediate molecule hapten. After washing, an anti-fade solution containing
incorporated into a probe) binding to DNA bases, which DAPI (4’, 6-diamidino-2-phenylindole) is applied to the
eventually evolved into fluorescence in situ hybridization slide, and a coverslip must be added. DAPI is a fluorescent
(FISH). FISH increased the resolution at which chromo- stain used extensively in fluorescence microscopy. FISH
some rearrangements could be identified at submicroscopic signals are typically observed using epifluorescence micro-
Riegel 197

scopes with specific filters for identifying fluorochromes probes, chromosome-arm painting probes, and repetitive
(Marcus, 1988; Reichman, 2000)), a charge-coupled device centromeric, subtelomeric and locus-specific probes) is
(CCD) camera captures the image and the fluorescent sig- available for the detection of certain constitutional and ac-
nals are subsequently quantified (Hiraoka et al., 1987). The quired chromosomal abnormalities. Nevertheless, FISH
resulting images can be analyzed using commercially avai- probes can be generated through chromosome flow sorting
lable systems. (Pinkel et al., 1988) or microdissection (Meltzer et al.,
Together with the development of standard FISH 1992) using universal degenerate oligonucleotide-primed
(Pinkel et al., 1986a,b), more sensitive FISH-based tech- PCR (DOP-PCR) (Telenius et al., 1992).
niques were gradually developed, and several digital imag- FISH is a flexible technique that has driven the fur-
ing systems were introduced for FISH image acquisition, ther development of other cytogenetic techniques. There
image pre-processing and digital image analysis. FISH pro- are multiple approaches using FISH-based methods for dif-
vides the option for the simultaneous use of one or more ferent applications, e.g., reverse-FISH (Carter et al., 1992),
DNA probes, and these probes can be distinguished after la- fiber-FISH (Florijn et al., 1995; Heiskanen et al., 1995),
beling with different colors or color combinations. The (M-FISH multicolor FISH) (Speicher et al., 1996), SKY
probes primarily determine the resolution of these molecu- (spectral karyotyping FISH) (Schröck et al., 1996), flow-
lar cytogenetic techniques and can be classified according FISH (Rufer et al., 1998), Q-FISH (quantitative FISH)
to the pattern of detected DNA sequences. Many types of (Martens et al., 1998), COBRA-FISH (combined binary ra-
probes can be used for FISH (Figure 2). Currently, a range tio labeling FISH) (Tanke et al., 1999), cenM-FISH
of commercial probes (e.g., whole-chromosome painting (centromere-specific M-FISH) (Nietzel et al., 2001), pod-

Figure 2 - FISH with different types of probes and partial metaphases. (a) Whole chromosome 21 painting; (b) partial chromosome painting probe for the
long arm of chromosome 9; (c) locus-specific probe for chromosome 4p16.3 (red) and Alfa satellite probe 4p11-q11(green); (d) subtelomeric probe for
the short arm (red) and long arm (green) of chromosome 1; (e) human telomeric probes; and (f) Interphase-FISH with locus-specific SRY (sex-
determining region Y) probe located in Yp11.31(red) and control probes for the X centromere (DXZ1) (blue) and for the heterochromatic block at Yq12
(green).
198 Human molecular cytogenetics

FISH (parental origin determination FISH) (Weise et al., technique was initially introduced to study chromosomal
2008), (heterochromatin-M-FISH) (Bucksch et al., 2012) abnormalities that occur in solid tumors and other malig-
and other modified FISH approaches. If modified, several nancies (Kallioniemi et al., 1992). Chromosomal CGH is
FISH techniques can also be applied to interphase cells based on quantitative two-color FISH and overcomes the
(interphase FISH) (Vorsanova et al., 2010), which confers problems of tissue culture failure and artifacts because this
the advantages of FISH for the visualization of DNA method is based on using tumor DNA extracted directly
probes in interphase nuclei (Cremer et al., 1986). The limi- from either fresh or archival tumor tissue (Kallioniemi,
tation of standard FISH, however, is that it is not possible to 2008). The major advantage of CGH over standard FISH
simultaneously detect all of the chromosomes in the entire techniques is that only the DNA from the tumor cells is
genome. needed for analysis, avoiding the difficulties of obtaining
COBRA-FISH, M-FISH, and SKY are the most ad- metaphase chromosomes with good morphology and reso-
vanced FISH-based approaches, and these approaches fa- lution for the analysis. In CGH, total genomic DNA ob-
cilitate the simultaneous visualization and detection of all tained from control cells and test samples is differentially
human and non-human chromosomes through color karyo- labeled using green (fluorescein isothiocyanate, FITC) and
typing. The simultaneous staining of each of the 24 human red (Texas red) fluorescent dyes, denatured, co-precipitated
chromosomes with a different color involves the use of in the presence of blocking DNA to suppress repetitive se-
whole-chromosome painting (WCP) probes, and all three quences and subsequently co-hybridized to normal meta-
of these FISH techniques use similar probe sets. Four to phase chromosomes. Due to the simultaneous hybridiza-
seven different fluorescence dyes can be used to label the tion to normal denatured metaphase chromosome spreads,
WCP probes, and the chromosomes are counterstained with there is competition for DNA hybridization to homologous
DAPI. The required 24 color combinations can be achieved sites. After hybridization and washing, the metaphase
through combinatorial or ratio labeling. The most impor- spreads are observed under a fluorescent microscope, and
tant aspect of these techniques is the acquisition and mea- image analysis is performed using image analysis software.
surement of the complete emission spectra between 400 The resulting fluorescence intensities of the test and refer-
and 800 nm, rendering a unique image that contains spe- ence hybridizations are digitally quantified along the length
cific spectral information for each image point. The result- of each chromosome. Chromosomal regions equally repre-
ing chromosome classification is performed automatically sented in both the test and reference samples appear yellow
using commercial software, and the DAPI image is also because of the presence of an identical amount of red and
used to complement the analysis with chromosome band- green dye, while regions with copy number loss are red and
ing information (Schröck et al., 2006). A high-resolution have a ratio below one (Figure 3a).
molecular cytogenetic technique for the analysis of meta- Although chromosomal CGH has increased the po-
phase chromosomes, called multicolor banding (MCB), has tential for identifying new chromosomal abnormalities, this
been proposed, which involves the microdissection of chro- technique is time consuming and does not significantly
mosomal loci to obtain a set of probes that produce improve resolution (> 3 Mb) compared with routine G-ban-
multicolor pseudo-G-banding (Liehr et al., 2002). ding chromosome analysis. More recently, the develop-
For either standard or advanced FISH methods, the ment of array-based CGH (array-CGH) approaches involv-
preparations should be analyzed using a well maintained ing the substitution of metaphase chromosomes with DNA
and calibrated fluorescence microscope equipped with the sequences adhered onto glass slides has increased the reso-
optical filter sets appropriate for the fluorochromes used lution for detecting copy number changes in the human ge-
and an image-recording system. The development of nu- nome, leading to more detailed information on genomic
merous FISH protocols and multiple approaches is the re- gains and losses (Figure 3b). Among all of the recent ad-
sult of the efforts of many diagnostic and research scientists vances in techniques for examining chromosomes, array-
from different research groups worldwide. These tech- CGH technology has been suggested as a technique that
niques have been continuously improved, and it is not pos- will gradually replace classical cytogenetics in clinical di-
sible to cover every modification of FISH in this agnosis. The fundamental principle of array-CGH is essen-
manuscript. Detailed FISH protocols and applications are tially the same as that in CGH. Indeed, the process involves
described elsewhere (Liehr, 2009). comparative genomic hybridization using an array rather
than a metaphase spread as the substrate (Solinas-Toldo et
al., 1997; Pinkel et al., 1998).
Comparative Genomic Hybridization (CGH) and
Array-based CGH The actual microarray comprises thousands of spots
of reference DNA sequences applied in a precisely gridded
The comparative genomic hybridization (CGH) tech- manner on the slide. The initial arrayed DNA segments
nique is an efficient approach to genome-wide screening could be larger (~150 kb) human DNA segments inserted
for chromosomal copy number changes (gains/duplications into a bacterial artificial chromosome (BAC clones) or bac-
and losses/deletions) within a single experiment, and this terial/P1-derived artificial chromosomes (PAC clones)
Riegel 199

Figure 3 - Comparative Genomic Hybridization. (A) Conventional CGH analysis: a mixture of test DNA from a patient and a normal reference DNA la-
beled with different fluorochromes are hybridized to normal chromosome spreads (top panel). The left panel illustrates the hybridization pattern of chro-
mosome 13. The interstitial segment of q-arm appears red, which indicates a loss of the region indicating rev ish dim (13q21q31). The right panel shows a
graph of the ratio profiles of chromosome 13. The black line represents the balanced fluorescence intensities, and the red line is the threshold for loss, and
the green line is the threshold for a gain of material. (B) Chromosome 4 array-CGH profile of a test DNA and a reference DNA. The figure shows a copy
number loss corresponding to the segment of 4p16.3-p15.33 in a genomic segment with the median log2 ratio shifted to -1.0. The lower panel shows the
4p16.3-p15.33 region with the deletion segment and the genes present in this region.

(Snijders et al., 2001; Fiegler et al., 2003; Chung et al., The number, size and distribution of the DNA seg-
2004; Ishkanian et al., 2004). As the resolution of the array ments on the glass slide determine the array resolution, but
yields improves, shorter sequences have been used as tar- commonly, the higher the number of DNA fragments, the
gets, including smaller cDNA fragments (Pollack et al., higher the resolution. According to Balliff et al. (2006) and
1999), PCR products (Mantripragada et al., 2004) and Cheung et al. (2007), array-CGH also has increased the
oligonucleotides (Rouillard et al., 2002). Furthermore, ar- sensitivity for detecting cell lines with chromosomal abnor-
ray-CGH provides resolution at the nucleotide level. Sin- malities in peripheral blood, as chromosomal abnormalities
gle-nucleotide polymorphism arrays (SNP arrays) have the are typically detected in only 5-7% of cells. Currently, there
highest resolution (5-10 kb) of all of the available ar- are several different commercially available diagnostic
ray-based platforms (see Le Scouarnec and Gribble, 2012).
DNA microarray platforms comparing thousands of DNA
The co-hybridization of the test and reference DNAs is not
sequences from a patient sample with reference (control)
required because the test DNA can hybridize directly to the
DNA samples or control datasets to detect chromosomal
SNP array. In addition to CNVs, the genotype information
obtained from SNP arrays enables the detection of stretches CNVs. A common limitation of SNP and CGH arrays is the
of homozygosity and thus the identification of recessive inability to identify balanced translocations and inversions.
disease genes, mosaic aneuploidy or uniparental disomy Recently, a modified array protocol, called trans-
(UPD) (de Leeuw et al., 2012). While only SNP arrays en- location CGH (tCGH), was developed to address recurrent
able the detection of copy number-neutral regions in the ab- translocation breakpoints in hematological neoplasms.
sence of heterozygosity (AOH), these arrays have limited Prior to the hybridization step in the array procedure, a lin-
ability to detect single-exon copy CNVs due to the distribu- ear PCR amplification is performed across the known re-
tion of SNPs across the genome. Combining both array- current translocation breakpoints in hematological neo-
CGH and SNP genotyping in a single platform optimizes plasms. Thus, it is possible to detect copy number changes
the clinical diagnostic capability, offering the simultaneous and known recurrent translocations near or at the break-
detection of copy number neutral and small intragenic copy points (Greisman et al., 2011). Custom-made commercial
number changes (Wiszniewska et al., 2014). arrays that use general standard protocols can also be or-
200 Human molecular cytogenetics

dered. Detailed information on the protocols and references microdeletion and microduplication syndromes (Riegel
is available elsewhere (Banerjee and Shah, 2013). and coworkers, unpublished data). However, FISH has lim-
itations in the detection of known microdeletion syn-
FISH Applications in Pre- and Postnatal dromes. Occasionally, patients with small and unusual
Diagnostics and Research deletions might escape detection, depending on the speci-
ficity of the fluorescent probe. Moreover, cases with gene
Several decades ago, molecular methods were intro- or imprinting mutations, occurring in some microdeletion
duced into cytogenetic studies, facilitating the development syndromes, e.g., Angelman syndrome (AS), Prader-Willi
of new applications, many of which were used diagnosti- syndrome (PWS), Sotos syndrome (SoS), Miller-Diecker
cally or as prognostic tools in medicine. Furthermore, mo- syndrome (MDS), Smith-Magenis syndrome (SMS) and
lecular cytogenetic approaches have also become indis- Rubinstein-Taybi syndrome (RTS), cannot be detected
pensable for a range of research purposes. The use of through FISH. The analysis of telomeres using FISH tech-
molecular techniques in cytogenetic studies is increasing, niques has been conducted in cancer and aging research
and the many variations, adaptations and specifications (telomere biology); however, due to the lack of specificity
make it challenging to cover all of the possible applications. of the DNA probes (TTAGGG repetitive sequence motifs),
Since the introduction of FISH in the late 1980s, there has this technique is poorly applicable for diagnosis (Aubert
been a tremendous increase in the number of studies using and Lansdorp, 2008). Multicolor FISH approaches have
molecular approaches in cytogenetics to detect chromo- been most valuable for cancer cytogenetics, but these meth-
somal abnormalities and evaluate CNVs in the human ge- ods have also been applied to diagnose constitutional chro-
nome. FISH offers numerous possibilities for studying ei- mosomal abnormalities (Liehr et al., 2004) and define
ther the whole genome or specific genomic loci (regions), translocations and marker chromosomes in complex karyo-
and this technique has been widely used to detect aneu- types (Kearney, 2006).
ploidies and recurrent chromosomal abnormalities in pre-
implantation genetic, prenatal, and postnatal diagnoses and Applications of CGH Analysis
cancer cytogenetics. Moreover, the application of FISH has
long been demonstrated as extremely valuable for studying Although CGH has primarily been applied to study
chromosomal and genome organization, evolution and solid tumors, this technique has also used to study leukemia
variations in health and disease (see Geurts and de Jong and lymphoma (Kallioniemi et al., 1992; Forozan et al.,
2013; McNamara et al., 2014; Pita et al., 2014). 1997; Gebhart, 2004; Carless, 2009). However, given that
A significant advantage of FISH is that it can be ap- CNVs are associated with many conditions, ranging from
plied in non-dividing cells, thereby facilitating the direct in- cancer to developmental abnormalities, CGH has also been
vestigation of chromosomes in cytological preparations applied to identify constitutional chromosomal abnormali-
and tissue sections. Classical cytogenetic analysis depends ties in clinical samples (Daniely et al., 1998; Lestou et al.,
on cells undergoing mitosis to obtain metaphase chromo- 1999; Kirchhoff et al., 2001; Ness et al., 2002; Schou et al.,
some spreads. Therefore, cells must be cultured in vitro ei- 2009). Several reports have demonstrated the use of either
ther as a short- or long-term culture. Thus, interphase FISH standard CGH or array-CGH to detect chromosomal abnor-
on uncultured amnion cells has become a useful method for malities in single cells of pre-implantation embryos (Wells
the rapid and early diagnosis of the most common chromo- and Delhanty, 2000; Le Caignec et al., 2006; Harton et al.,
some disorders (trisomies 21, 13, 18 and sex chromosome 2013).
aneuploidies) in fetal cells (Eiben et al., 1998). For prenatal Array-CGH was initially applied to identify chromo-
aneuploidy screening using uncultured amniocytes, no somal imbalances through the detection of CNVs in tumors
time-consuming cell culture is required, and the results can to distinguish candidate genes involved in the pathogenesis
be obtained within 24-48 hours. Three satellite centromeric of cancer (Cai et al., 2002; Albertson and Pinkel, 2003). In
probes for chromosomes X, Y and 18 and two locus- clinical diagnostics, both oligonucleotide array-CGH and
specific probes for the 13q14 and 21q22.13 regions are the SNP genotyping have been demonstrated as powerful ge-
most commonly applied. Interphase FISH in prenatal diag- nomic technologies for evaluating idiopathic mental retar-
nosis is a quick, accurate, sensitive and relatively specific dation (MR) (also referred to as developmental delay (DD),
method to detect aneuploidies in samples of uncultured intellectual disability (ID) or learning difficulty), associ-
chorionic villus (Rosner et al., 2013) and amniotic fluid ated congenital abnormalities (MCA), autistic spectrum
cells (Stumm et al., 2006). disorders (ASDs), schizophrenia and other neuropsychiat-
Using site-specific DNA probes (YACs, BACs, ric disorders. Furthermore, the introduction of genome-
PACs, and cosmids), FISH is typically applied for mapping wide array platforms facilitated the detection of chromo-
chromosomal regions with located breakpoints (Liehr, somal abnormalities consistent with genetic syndromes at
2009). In addition, using locus-specific probes, FISH has earlier ages, when only a few clinical findings might be
also been used to confirm clinical diagnoses of known present.
Riegel 201

CNVs are DNA segments that present a variable copy patients with schizophrenia. This rate only considers the
number compared with a reference genome, which has the currently known CNVs. Thus, it is likely that many more
typical copy number of N = 2 (Feuk et al., 2006). In 2004, unique CNVs with major effects exist, similarly to ASD. In
two studies employing array-based platforms revealed that a small fraction of patients with schizophrenia, the alleles
CNVs exist in many large DNA genomic segments be- with CNVs are likely the strongest factors contributing to
tween normal human individuals, suggesting that these the pathogenesis of the disease (Stefansson et al., 2014).
variations are fairly common and might represent polymor- Recently, Nicholl et al. (2014) reported the frequency
phic variations and a significant source of genetic variation of pathogenic chromosomal microdeletions and microdu-
(Iafrate et al., 2004; Sebat et al., 2004). Furthermore, the plications in a large group of referred patients with devel-
examination of the genomic content of CNVs revealed that opmental delay (DD), intellectual disability (ID) or autism
these genomic regions include many functional genes in- spectrum disorders (ASD), and these authors provided a ge-
volved in the regulation of cell growth and metabolism netic diagnostic service. The first tier testing was applied
(Iafrate, 2004), implicating CNVs in human traits, disease using a standardized oligo-array CGH platform. The fol-
and evolution. Since that time, many additional studies us- lowing detection rates, excluding the CNVs of uncertain
ing a multitude of different high-resolution genome- significance, were observed: DD (13.0%), ID (15.6%),
analysis platforms have advanced our knowledge regarding ASD (2.3%), ASD with DD (8.2%), ASD with ID (12.7%)
CNVs. and unexplained epilepsy with DD, ID and ASD (10.9%).
Since Vissers et al. (2003) published the first report Greater diagnostic sensitivity reflects the routine applica-
on detecting constitutional submicroscopic imbalances us- tion of array CGH, compared with previously used conven-
ing array-based techniques in a series of patients with tional cytogenetics; according to Nicholl et al. (2014), the
ID/MCA, the results of many more array-based studies greater diagnostic sensitivity outweighs the interpretative
have been published. Array-based genome investigations issues arising from the detection of CNVs of uncertain sig-
have been demonstrated to detect pathogenic imbalances in nificance.
approximately 14-18% of consecutive ID/MCA cases re- Microarray approaches are increasingly used in pre-
ferred for analysis. The rate differences might reflect differ- natal settings in pregnancies with ultrasound anomalies and
ences in the resolutions of the array platforms used, the pregnancies referred for other reasons. However, chal-
criteria for patient selection and the interpretation of the lenges in interpreting the results, quality control and ethical
clinical relevance of the CNVs detected. Most of these issues have delayed the use of microarray approaches in
CNVs are deletions and duplications that arise de novo, ei- prenatal care compared with postnatal diagnoses (Rickman
ther as unique or recurrent events (Hochstenbach et al., et al., 2005; Vetro et al., 2012). Numerous case series and
2011). The increasing number of laboratories worldwide case reports have since been published on the application of
applying array-based methods for the diagnosis of patients array-CGH in prenatal settings (Brady and Vermeesch,
with multiple congenital abnormalities has increased the 2012; Brady et al., 2013; Evangelidou et al., 2013). Ar-
detection of human genomic imbalances and led to the ray-CGH increases the diagnostic yield for detecting addi-
identification of a number of diseases caused by chromo- tional genomic imbalances 1-5% compared with normal
somal microdeletions and microduplications. In recent karyotyping, depending on the reference source (ACOG
years, common and newer microdeletion and microdu- Committee, 2009; Hillman et al., 2011; Lichtenbelt et al.,
plication syndromes associated with a variety of pheno- 2011).
types have been revisited (Schinzel et al., 2013; Riegel and In hematologic and oncologic disorders, the imple-
coworkers, unpublished data;) and recognized (Deak et al., mentation of array-based chromosome analysis has been
2011; Rafati et al., 2012; Vissers and Stankiewicz, 2012; critical. The complexity of cancer cells requires a sensitive
Weise et al., 2012; Shimizu et al., 2013). technique that facilitates the detection of small genomic
The use of array-CGH as a genetic test in selected changes in a mixed cell population and segmental regions
sporadic ASD patients has shown that non-syndromic, de of homozygosity. However, recurrent balanced genomic
novo CNVs occur in ~7.5% of boys and ~12% of girls. De aberrations with important prognostic value in cancer
novo deletions CNVs in female patients tend to be larger might be not detected through array-based analyses. Be-
than in male patients and contain a higher number of pro- cause array-CGH is based on the principle of CNV detec-
tein-coding genes (Sanders et al., 2011). According to tion, this technique is limited by an inability to identify
Hochstenbach et al. (2011), these findings suggest that balanced translocations and inversions. Nevertheless, ar-
women are more resistant than men to developing ASD and rays have been previously demonstrated as clinically essen-
are less likely to be diagnosed with ASD or both. In syn- tial for identifying novel genomic abnormalities that escape
dromic ASD cases, the chance of finding a causal CNV is detection using current diagnostic methodologies in a num-
nearly 25%. Based on recurrent microdeletions and micro- ber of hematological diseases, such as chronic lymphocytic
duplications identifiable on array-based platforms, a con- leukemia (CLL), myelodysplastic syndrome (MDS), multi-
tributing CNV can be expected in approximately 5% of ple myeloma (MM), acute lymphoblastic leukemia (ALL),
202 Human molecular cytogenetics

acute myeloid leukemia (AML) and chronic myelomo- combinations of variants lead to varying degrees of patho-
nocytic leukemia (CMML) (Shao et al., 2010; Simons et genicity. Factors that influence the pathogenicity of CNVs
al., 2012). Moreover, the identification and accurate geno- and an evidence-based classification for the clinical inter-
mic mapping of genomic alterations in hematological pretation of CNVs have been discussed and proposed (Lee
malignances in a preclinical stage have shown that it is pos- et al., 2007; Hehir-Kwa et al., 2010; Miller et al., 2010;
sible to refine the current risk stratification of patients, and Gijsbers et al., 2011; de Leeuw et al., 2012; Riggs et al.,
this technique might eventually contribute to the develop- 2012; Liehr, 2014). Online resources and public databases
ment of enhanced treatment modalities (van der Veken and have been developed and are utilized by the scientific and
Buijs, 2011; Simons et al., 2012). biomedical community, which has been encouraged to sub-
The detection of common and rare CNVs using ar- mit cases to the databases to provide data on the test results
ray-based platforms has generated questions concerning (Vulto-van Silfhout et al., 2013).
the origin and molecular mechanisms leading to recurrent Common strategies have been proposed to help inter-
and non-recurrent CNVs (Lupski and Stankiewicz 2005; pret CNV findings, and no universal criteria have been es-
Currall et al., 2013; Dittwald et al., 2013; Sun et al., 2013) tablished thus far. Most laboratories classify the various
and the phenotypic effects of CNVs and recurrence risks CNVs into different categories using some or all of the
(Girirajan et al., 2012; Priest et al., 2012; Boone et al., CNV classifications: benign CNV or normal genomic vari-
2013). Recombination-based mechanisms, i.e., non-allelic ant; benign CNV; CNV with uncertain clinical relevance or
homologous recombination (NAHR), non-homologous variants of uncertain significance (VOUS); and CNV with
end joining (NHEJ) (Lupski and Stankiewicz, 2005) and potential clinical relevance or pathogenic variants. When
retrotransposition (Kazazian Jr and Moran, 1998; Xing et array-CGH was initially used, all identified CNVs were
al., 2009), have been implicated in genomic rearrange- generally reported. In recent years, the trend towards stan-
ments and the formation of CNVs. A replication-based dardizing the reporting among laboratories worldwide, and
mechanism, fork stalling and template switching (FoSTeS) the current tendency is to report only potentially meaning-
might account for the complex genomic rearrangements ful CNVs. Nevertheless, the array platform used and the re-
that cannot be readily explained through NAHR, NHEJ or porting criteria might vary between individual laboratories.
retrotransposition (Lee et al., 2007; Perry et al., 2008; Arlt Different laboratories might also use different methods to
et al., 2012). CNVs represent an important component of confirm the array findings (e.g., FISH, multiplex ligation-
genetic variation and have been described as a major con- dependent probe amplification (MLPA), Quantitative Flu-
tributor to phenotype diversity and disease (Girirajan and orescence Polymerase Chain Reaction (QF-PCR), and a
Eichler, 2010; Arlt et al., 2011; Cooper et al., 2011; Giri- second array-CGH).
rajan et al., 2011; Girirajan, 2013). When interpreting and classifying CNVs, it is essen-
tial to distinguish gains from losses because the potential
Interpretation of CNVs clinical consequences might significantly differ. Further-
The widespread use of array-CGH has revealed that a more, it is essential to compare gains with gains and losses
large proportion of the human genome contains regions of with losses (Vermeesch et al., 2007; Conrad et al., 2010;
copy number variability, and distinguishing between Vermeesch et al., 2012). de Leeuw et al. (2012) summa-
pathogenic and benign gains and losses has been challeng- rized the characteristics of the most commonly used
ing. Although array-CGH technology has been well devel- Internet databases and resources and proposed a general in-
oped and there are numerous algorithms available for terpretation strategy that can be used for comparative hy-
estimating copy number (McDonnell et al., 2013), the reso- bridization, comparative intensity and genotype-based
lution of the array platforms used in molecular cytogenetics array data. Some of the available online databases associ-
and our understanding of the clinical effects of CNVs are ated with chromosome abnormalities and variants are listed
still improving. Recurrent CNVs can occur in both patients below (as of January 2014):
and healthy individuals, and frequently, more than one Centre for the Development and Evaluation of Com-
unique CNV is identified in a patient. A given copy number plex Interventions for Public Health Improvement
change with a high penetrance pathogenic might reduce or (DECIPHER) project: http://decipher.sanger.ac.uk.
aggravate the clinical phenotype in the presence of other The Chromosome Anomaly Collection:
CNVs/SNPs. For example, Girirajan et al. (2010) demon- http://www.ngrl.org.uk/wessex/collection/.
strated that the 16p11.2 microdeletion predisposes individ- Chromosomal Variation in Man Online Database:
uals to neuropsychiatric phenotypes as a single event and http://www.wiley.com/legacy/products/sub-
aggravates neurodevelopmental phenotypes in association ject/life/borgaonkar/access.html.
with other large deletions or duplications within the ge- Cytogenetic Data Analysis System (CyDAS):
nome of an individual. http://www.cydas.org/.
The large quantity of clinical and cytogenetic data Database of genomic structural variation (bdVar):
available in open access databases can help decipher which http://www.ncbi.nlm.nih.gov/dbvar/.
Riegel 203

Ensembl: www.ensembl.org/. demonstrated as powerful genomic technologies to evalu-

European Cytogeneticists Association Register of ate DD, MCAs and neuropsychiatric disorders. Differences
Unbalanced Chromosome Aberrations (ECARUCA): in the ability to detect genomic changes between these ar-
www.ecaruc.net. rays might constitute a challenge for laboratory managers,
The International Standards for Cytogenomic Arrays as the request to provide the best approach to detect under-
(ISCA) Consortium:https://www.iscaconsortium.org/in- lying genetic causes of diseases is increasing. In most
dex.php. cases, imbalances that are cytogenetically visible in size
Small supernumerary marker chromosomes: (several Mb) lead to severe clinical consequences and are
http://ssmc-tl.com/sSMC.html. responsible for specific syndromes or clinical features
(Schinzel, 2001). However, CNVs can be expected in every
Final Remarks individual on a chromosomal or molecular genetic level
(1000 Genomes Project Consortium et al., 2012). Thus, it is
The methods described herein provide information on
expected that the identification of variants of unknown
the human genome at different levels of resolution and have
clinical significance will significantly increase, particularly
shown potential for diagnostic and research purposes. The
as many individuals now have their entire genomes se-
resolution for studying chromosomes has improved from >
quenced (Bale et al., 2011; Palmer et al., 2014). Segmental
5 Mb (metaphase) to 50 kb-2 Mb (interphase) and 5-500 kb
chromosome regions that might be present in variable copy
(DNA fibers) and ultimately, to a single nucleotide. Molec-
numbers in the genome without phenotypic consequences
ular cytogenetics and array-based technologies facilitate
are constantly being identified (Barber, 2005; Liehr, 2012).
higher resolutions through genome-wide screening for sub-
microscopic genomic CNVs. However, to identify cyto- To date, the critical point has been to distinguish simi-
genetically visible CNVs (e.g., heterochromatin), low lar-looking benign imbalances from pathological imbal-
mosaicisms and balanced translocations, banding cyto- ances. To facilitate the interpretation and analysis of the
genetics has been demonstrated as useful. Cytogenetic test- information obtained using molecular cytogenetic ap-
ing in developed countries primarily uses array-CGH tech- proaches, widely available public databases have been de-
nology to detect novel or rare veloped and are constantly updated (e.g., CyDAS,
microdeletions/microduplications and has become the DECIPHER, ECARUCA, ISCA). Nevertheless, many
first-line test in the diagnostic investigation of individuals genomic imbalances are novel or extremely rare, making
with MCAs, DDs or unexplained IDs. Although the use of interpretation problematic and uncertain. Thus, further mo-
banding and FISH has gradually been replaced by ar- lecular cytogenetic screenings of large patient cohorts with
ray-based technologies in several laboratories, G-banding common phenotypic features contribute to the ongoing de-
remains the most commonly used approach worldwide to velopment of genotype-phenotype correlations, identifying
study the human genome. Moreover, the comparison of CNVs in dosage-sensitivity genes and defining their loca-
chromosome and array-based chromosome analyses has tions in the human genome. The use of whole-genome se-
demonstrated that chromosome analysis remains valuable quencing and whole-exome sequencing platforms has been
for detecting mosaicisms and to delineate chromosomal increasingly popular and powerful for genetic diagnosis
structural rearrangements (Bi et al., 2013). Evaluating the (Bick and Dimmock, 2011; Greisman et al., 2013; Johan-
use of conventional karyotypes or molecular approaches sen Taber et al., 2013; Rabbani et al., 2014). These methods
will likely require continuous evaluation, as questions re- might potentially be alternatives to the use of microarrays
garding how to achieve cost-effective diagnoses still re- in molecular cytogenetic laboratories. The technologies ap-
main in many clinical situations, e.g., rare chromosome plied to study genomic imbalances have been rapidly
breakage syndromes and low-risk pregnancies (van changing. Therefore, the comprehensive collection, organi-
Ravenswaaij-Arts, personal communication 2013). zation and maintenance of the raw genotype-phenotype
As the number of recognized genetic syndromes and data obtained through different approaches are major chal-
chromosomal abnormalities grows and as the clinical char- lenges.
acteristics of those syndromes overlap, it will be more diffi- The implementation and updating of national, re-
cult to precisely infer which syndrome affects an individual gional and international guidelines on the indications and
based only on the clinical examination. Currently, the de- interpretations of molecular cytogenetics results along with
tection of large numbers of CNVs using molecular cyto- clinical management to improve expertise and experience
genetic approaches in patients and healthy individuals has in clinical and laboratory praxis are necessary to improve
been considered a diagnostic pitfall due to interpretation scientific knowledge and medical care. In addition, the re-
difficulties. Most chromosomal abnormalities have clinical porting of molecular cytogenetic results is also another im-
effects; however, the number of instances in which geno- portant issue (ISCN. An International System for Human
mic changes are benign has increased, as the resolution of Cytogenetic Nomenclature, 2013). As new techniques are
chromosome analysis has also increased. In clinical diag- implemented in cytogenetic laboratories for clinical use,
nosis, both array-CGH and SNP genotyping have been additional provisions for reporting findings should be de-
204 Human molecular cytogenetics

veloped though international guidelines. The number of Bi W, Borgan C, Pursley AN, Hixson P, Shaw CA, Bacino CA,
chromosomal abnormalities and potential genomic rear- Lalani SR, Patel A, Stankiewicz P, Lupski JR, et al. (2013)
rangements in the human genome are likely unlimited. In Comparison of chromosome analysis and chromosomal
microarray analysis: What is the value of chromosome anal-
the last decade, the importance of both high-quality cyto-
ysis in today’s genomic array era? Genet Med 15:450-457.
genetics and genome sequencing for detecting and under-
Bick D and Dimmock D (2011) Whole exome and whole genome
standing the molecular mechanisms that lead to these sequencing. Curr Opin Pediatr 23:594-600.
chromosomal changes has been clear. Regardless of the de- Blakeslee AF and Avery AG (1937) Methods of inducing dou-
velopment of next-generation molecular techniques for bling of chromosomes in plants. J Hered 28:392-411.
identifying chromosomal imbalances and CNVs in the hu- Boone PM, Campbell IM, Baggett BC, Soens ZT, Rao MM,
man genome, the essential purpose of cytogenetics will re- Hixson PM, Patel A, Bi W, Cheung SW, Lalani SR, et al.
main the same: to study genomic organization and the (2013) Deletions of recessive disease genes: CNV contribu-
structure, function and evolution of chromosomes. tion to carrier states and disease-causing alleles. Genome
Res 23:1383-1394.
Brady PD and Vermeesch JR (2012) Genomic microarrays: A
Acknowledgments
technology overview. Prenat Diag 32:336-343.
The author would like to apologize to those col- Brady PD, Delle Chiaie B, Christenhusz G, Dierickx K, Van Den
leagues whose contributions to the field have been unwit- Bogaert K, Menten B, Janssens S, Defoort P, Roets E, Sleurs
tingly omitted and to the authors of relevant papers who E, et al. (2013) A prospective study of the clinical utility of
could not be cited because of space limitations. Unpub- prenatal chromosomal microarray analysis in fetuses with
lished results were generally not included except as person- ultrasound abnormalities and an exploration of a framework
for reporting unclassified variants and risk factors. Genet
ally observed by the author.
Med 2013 [Epub ahead of print].
Brambati B and Simoni G (1983) Diagnosis of fetal trisomy 21 in
References first trimester. Lancet 1:586.
1000 Genomes Project Consortium, Abecasis GR, Auton A, Bucksch M, Ziegler M, Kosayakova N, Mulatinho MV, Llerena Jr
Brooks LD, DePristo MA, Durbin RM, Handsaker RE, JC, Morlot S, Fischer W, Polityko AD, Kulpanovich AI,
Kang HM, Marth GT and McVean GA (2012) An integrated Petersen MB, et al. (2012) A new multicolor fluorescence in
map of genetic variation from 1,092 human genomes. Na- situ hybridization probe set directed against human hetero-
ture 491:56-65. chromatin: HCM-FISH. J Histochem Cytochem 60:530-
ACOG Committee (2009) Opinion No. 446: Array comparative 536.
genomic hybridization in prenatal diagnosis. Obstet Cai WW, Mao JH, Chow CW, Damani S, Balmain A and Bradley
Gynecol 114:1161-1163. A (2002) Genome-wide detection of chromosomal imbal-
Albertson DG and Pinkel D (2003) Genomic microarrays in hu- ances in tumors using BAC microarrays. Nat Biotechnol
man genetic disease and cancer. Hum Mol Genet 2 (Spec No 20:393-396.
2):R145-R152. Carless M (2009) Analysis of genomic aberrations using compar-
Arlt MF, Ozdemir AC, Birkeland SR, Lyons Jr RH, Glover TW ative genomic hybridization of metaphase chromosomes.
and Wilson TE (2011) Comparison of constitutional and Methods Mol Biol 523:177-202.
replication stress-induced genome structural variation by Carter NP, Ferguson-Smith MA, Perryman MT, Telenius H, Pel-
SNP array and mate-pair sequencing. Genetics 187:675-83. mear AH, Leversha MA, Glancy MT, Wood SL, Cook K,
Arlt MF, Wilson TE and Glover TW (2012) Replication stress and Dyson HM, et al. (1992) Reverse chromosome painting: A
mechanisms of CNV formation. Curr Opin Genet Dev method for the rapid analysis of aberrant chromosomes in
22:204-210. clinical cytogenetics. J Med Genet 29:299-307.
Arnold J (1879) Beobachtungen über Kernteilungen in den Zellen Caspersson T, Farber S, Foley GE, Kudynowski J, Modest EJ,
der Geschwülste. Virchows Arch Pathol Anat 78:279. Simonsson E, Wagh U and Zech L (1968) Chemical differ-
Aubert G and Lansdorp PM (2008) Telomeres and aging. Physiol entiation along metaphase chromosomes. Exp Cell Res
Ver 88:557-579. 49:219-222.
Bale S, Devisscher M, Van Criekinge W, Rehm HL, Decouttere F, Cheung SW, Shaw CA, Scott DA, Patel A, Sahoo T, Bacino CA,
Nussbaum R, Dunnen JT and Willems P (2011) Pursley A, Li J, Erickson R, Gropman AL, et al. (2007)
MutaDATABASE: A centralized and standardized DNA Microarray-based CGH detects chromosomal mosaicism
variation database. Nat Biotechnol 29:117-1188. not revealed by conventional cytogenetics. Am J Med Genet
Balliff BC, Rorem EA, Sundin K, Linicium M, Gaskin S, Cop- A 143:1679-1686.
pinger J, Kashork CD, Shaffer LG and Beijani BA (2006) Chung YJ, Jonkers J, Kitson H, Fiegler H, Humphray S, Scott C,
Detection of low-level mosaicism by array CGH in routine Hunt S, Yu Y, Nishijima I, Velds A, et al. (2004) A whole-
diagnostic specimens. Am J Med Genet A 140A:2757-2767. genome mouse BAC microarray with 1-Mb resolution for
Banerjee D and Shah SP (2013) Methods in Molecular Biology analysis of DNA copy number changes by array compara-
973: Array Comparative Genomic Hybridization (Protocols tive genomic hybridization. Genome Res 14:188-196.
and Applications). Humana Press, New Jersey, 382 pp. Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, Zhang Y,
Barber JC (2005) Directly transmitted unbalanced chromosome Aerts J, Andrews TD, Barnes C, Campbell P, et al. (2010)
abnormalities and euchromatic variants. J Med Genet Origins and functional impact of copy number variation in
42:609-629. the human genome. Nature 464:704-712.
Riegel 205

Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, Forozan F, Karhu R, Kononen J, Kallioniemi A and Kallioniemi
Williams C, Stalker H, Hamid R, Hannig V, et al. (2011) A OP (1997) Genome screening by comparative genomic hy-
copy number variation morbidity map of developmental de- bridization. Trends Genet 13:405-409.
lay. Nat Genet 43:838-846. Gall JG and Pardue ML (1969) Formation and detection of RNA-
Cremer T, Landegent J, Brückner A, Scholl HP, Schardin M, DNA hybrid molecules in cytological preparations. Proc
Hager HD, Devilee P, Pearson P and van der Ploeg M (1986) Natl Acad Sci USA 63:378-383.
Detection of chromosome aberrations in the human inter- Gartler SM (2006) The chromosome number in humans: A brief
phase nucleus by visualization of specific target DNAs with history. Nat Rev Genet 7:655-660.
radioactive and non-radioactive in situ hybridization tech- Gebhart E (2004) Comparative genomic hybridization (CGH):
niques: Diagnosis of trisomy 18 with probe L1.84. Hum Ten years of substantial progress in human solid tumor mo-
Genet 74:346-352. lecular cytogenetics. Cytogenet Genome Res 104:352-358.
Currall BB, Chiang C, Talkowski ME and Morton CC (2013) German JL and Bearn AG (1961) Asynchronous thymidine up-
Mechanisms for structural variation in the human genome. take by human chromosomes. J Clin Invest 40:1041-1042.
Curr Genet Med Rep 1:81-90. Geurts R and de Jong H (2013) Fluorescent In Situ Hybridization
Daniely M, Aviram-Goldring A, Barkai G and Goldman B (1998) (FISH) on pachytene chromosomes as a tool for genome
Detection of chromosomal aberration in fetuses arising from characterization. Methods Mol Biol 1069:15-24.
recurrent spontaneous abortion by comparative genomic hy- Gijsbers AC, Schoumans J and Ruivenkamp CA (2011) Interpre-
bridization. Hum Reprod 13:805-809. tation of array comparative genome hybridization data: A
de Leeuw N, Dijkhuizen T, Hehir-Kwa JY, Carter NP, Feuk L, major challenge. Cytogenet Genome Res 135:222-227.
Firth HV, Kuhn RM, Ledbetter DH, Martin CL, van Ravens- Girirajan S (2013) Genomic disorders: Complexity at multiple
waaij-Arts CM, et al. (2012) Diagnostic interpretation of ar- levels. Genome Med 29:5-43.
ray data using public databases and internet sources. Hum Girirajan S and Eichler EE (2010) Phenotypic variability and ge-
Mutat [Epub ahead of print]. netic susceptibility to genomic disorders. Hum Mol Genet
Deak KL, Horn SR and Rehder CW (2011) The evolving picture 19(R2):R176-R87.
of microdeletion/microduplication syndromes in the age of Girirajan S, Rosenfeld JA, Cooper GM, Antonacci F, Siswara P,
microarray analysis: Variable expressivity and genomic Itsara A, Vives L, Walsh T, McCarthy SE, Baker C, et al.
complexity. Clin Lab Med 4:543-564. (2010) A recurrent 16p12.1 microdeletion supports a two-hit
Dittwald P, Gambin T, Szafranski P, Li J, Amato S, Divon MY, model for severe developmental delay. Nat Genet 42:203-
Rodríguez Rojas LX, Elton LE, Scott DA, Schaaf CP, et al. 209.
(2013) P NAHR-mediated copy-number variants in a clini- Girirajan S, Campbell CD and Eichler EE (2011) Human copy
cal population: Mechanistic insights into both genomic dis- number variation and complex genetic disease. Annu Rev
orders and Mendelizing traits. Genome Res 23:1395-1409. Genet 45:203-226.
Eagle H (1955) Nutrition Needs of Mammalian Cells in Tissue Girirajan S, Rosenfeld JA, Coe BP, Parikh S, Friedman N, Gol-
Culture. Science 122:501-504. dstein A, Filipink RA, McConnell JS, Angle B, Meschino
Eiben B, Hammans W, Goebel R and Epplen JT (1998) Ein neuer WS, et al. (2012) Phenotypic heterogeneity of genomic dis-
Schnelltest (FISH) zur pränatalen Diagnostik der häufigsten orders and rare copy-number variants. N Engl J Med
Chromosomenaberrationen - welche Bedeutung hat er für 367:1321-1331.
die Praxis? Dtsch Med Wochenschr 123:55-57. Greisman HA, Hoffman NG and Yi HS (2011) Rapid high-
Evangelidou P, Alexandrou A, Moutafi M, Ioannides M, Anto- resolution mapping of balanced chromosomal rearrange-
niou P, Koumbaris G, Kallikas I, Velissariou V, Sismani C ments on tiling CGH arrays. J Mol Diagn 13:621-633.
and Patsalis PC (2013) Implementation of high resolution Greisman HA, Hoffman NG, Yi HS. Grody WW, Thompson BH
whole genome array CGH in the prenatal clinical setting: and Hudgins L (2013) Whole-exome/genome sequencing
Advantages, challenges, and review of the literature. Bio- and genomics. Pediatrics 132(Suppl 3):S211-S215.
med Res Int 2013:e346762. Hahnemann N (1974) Early prenatal diagnosis: A study of biopsy
Feuk L, Carson AR and Scherer SW (2006) Structural variation in techniques and cell culturing from extraembryonic mem-
the human genome. Nat Rev Genet 7:85-97. branes. Clin Genet 6:294-306.
Fiegler H, Gribble SM, Burford DC, Carr P, Prigmore E, Porter Hansemann D (1890) Über asymmetrische Zellteilung in
KM, Clegg S, Crolla JA, Dennis NR, Jacobs P, et al. (2003) Epithelkrebsen und deren biologische Bedeutung. Arch
Array painting: A method for the rapid analysis of aberrant Pathol Anat 119:299-326.
chromosomes using DNA microarrays. J Med Genet Harton GL, Munné S, Surrey M, Grifo J, Kaplan B, McCulloh
40:664-670. DH, Griffin DK, Wells D and PGD Practitioners Group
Flemming W (1882) Beiträge zur Kenntnis der Zelle und ihrer (2013) Diminished effect of maternal age on implantation
Lebenserscheinungen III. Arch Mikrosk Anat 20:1. after preimplantation genetic diagnosis with array compara-
Florijn RJ, Bonden LA, Vrolijk H, Wiegant J, Vaandrager JW, tive genomic hybridization. Fertil Steril 100:1695-1703.
Baas F, den Dunnen JT, Tanke HJ, van Ommen GJ and Raap Hehir-Kwa JY, Wieskamp N, Webber C, Pfundt R, Brunner HG,
AK (1995) High-resolution DNA Fiber-FISH for genomic Gilissen C, de Vries BB, Ponting CP and Veltman JA (2010)
DNA mapping and colour bar-coding of large genes. Hum Accurate distinction of pathogenic from benign CNVs in
Mol Genet 4:831-836. mental retardation. PLoS Comput Biol 6:e1000752.
Ford CE, Miller OJ, Polani PE, de Almeida JC and Briggs JH Heiskanen M, Hellsten E, Kallioniemi OP, Mäkelä TP, Alitalo K,
(1959) A sex-chromosome anomaly in a case of gonadal Peltonen L and Palotie A (1995) Visual mapping by fi-
dysgenesis (Turner’s syndrome). Lancet 1:711-713. ber-FISH. Genomics 30:31-36.
206 Human molecular cytogenetics

Hillman SC, Pretlove S, Coomarasamy A, McMullan DJ, Davison Lejeune J, Gautier M and Turpin MR (1959) Etude des chromo-
EV, Maher ER and Kilby MD (2011) Additional informa- somessomatiques de neuf enfants mongoliens. C R Acad Sci
tion from array comparative genomic hybridization technol- (Paris) 248:1721-1722.
ogy over conventional karyotyping in prenatal diagnosis: A Lestou VS, Lomax BL, Barrett IJ and Kalousek DK (1999) Scre-
systematic review and meta-analysis. Ultrasound Obstet ening of human placentas for chromosomal mosaicism using
Gynecol 37:6-14. comparative genomic hybridization. Teratology 59:325-
Hiraoka Y, Sedat JW and Agard DA (1987) The use of a charge- 330.
coupled device for quantitative optical microscopy of bio- Levan A (1938) The effect of colchicine on root mitosis in Allium.
logical structures. Science 238:36-41. Heredity 24:471-486.
Hochstenbach R, Buizer-Voskamp JE, Vorstman JA and Ophoff Lichtenbelt KD, Knoers NV and Schuring-Blom GH (2011) From
RA (2011) Genome arrays for the detection of copy number karyotyping to array-CGH in prenatal diagnosis. Cytogenet
variations in idiopathic mental retardation, idiopathic gener- Genome Res 135:241-250.
alized epilepsy and neuropsychiatric disorders: Lessons for Liehr T (2009) Fluorescence In Situ Hybridization (FISH) Appli-
diagnostic workflow and research. Cytogenet Genome Res cation Guide. Springer, Berlin, 452 pp.
135:174-202. Liehr T (2012) Small Supernumerary Marker Chromosomes
Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi (SSMC). A Guide for Human Geneticists and Clinicians.
Y, Scherer SW and Lee C (2004) Detection of large-scale Springer, Heidelberg, 220 pp .
variation in the human genome. Nat Genet 36:949-51. Liehr T (2014) Benign & Pathological Chromosomal Imbalances.
ISCN (2013) An International System for Human Cytogenetic Microscopic and Submicroscopic Copy Number Variations
Nomenclature. Karger, Basel, 140 pp. (CNVs) in Genetics and Counseling. Elsevier, London, 232
Ishkanian AS, Malloff CA, Watson SK, DeLeeuw RJ, Chi B, Coe pp.
BP, Snijders A, Albertson DG, Pinkel D, Marra MA, et al. Liehr T, Heller A, Starke H, Rubtsov N, Trifonov V, Mrasek K,
(2004) A tiling resolution DNA microarray with complete Weise A, Kuechler A and Claussen U (2002) Microdis-
coverage of the human genome. Nat Genet 36:299-303. section based high resolution multicolor banding for all 24
Jacobs PA and Strong JA (1959) A case of human intersexuality human chromosomes. Int J Mol Med 9:335-339.
having a possible XXY sex-determining mechanism. Nature Liehr T, Starke H, Weise A, Lehrer H and Claussen U (2004)
183:302-303. Multicolor FISH probe sets and their applications. Histol
Johansen Taber KA, Dickinson BD and Wilson M (2013) The Histopathol 19:229-237.
promise and challenges of next-generation genome sequenc- Lupski JR and Stankiewicz P (2005) Genomic disorders: Molecu-
ing for clinical care. JAMA Intern Med 174:275-280. lar mechanisms for rearrangements and conveyed pheno-
types. PLoS Genet 1:e49.
John HA, Birnstiel ML and Jones KW (1969) RNA-DNA hybrids
at the cytological level. Nature 223:582-587. Mantripragada KK, Tapia-Páez I, Blennow E, Nilsson P, Wedell
A and Dumanski JP (2004) DNA copy-number analysis of
Kallioniemi A (2008) CGH microarrays and cancer. Curr Opin
the 22q11 deletion-syndrome region using array-CGH with
Biotechnol 19:36-40.
genomic and PCR-based targets. Int J Mol Med 13:273-279.
Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW,
Marcus DA (1988) High-performance optical filters for fluores-
Waldman F and Pinkel D (1992) Comparative genomic hy-
cence analysis. Cell Motil Cytoskeleton 10:62-70.
bridization for molecular cytogenetic analysis of solid tu-
Martens UM, Zijlmans JM, Poon SS, Dragowska W, Yui J,
mors. Science 258:818-821.
Chavez EA, Ward RK and Lansdorp PM (1998) Short telo-
Kazazian Jr HH and Moran JV (1998) The impact of L1 retro- meres on human chromosome 17p. Nat Genet 18:76-80.
transposons on the human genome. Nat Genet 19:19-24. Matthey R (1949) Les Chromosomes des Vertébrés. Librairie de
Kearney L (2006) Multiplex-FISH (M-FISH): Technique, devel- I'Universite, F. Rouge-Lausanne, 356 pp.
opments and applications. Cytogenet Genome Res McDonnell SK, Riska SM, Klee EW, Thorland EC, Kay NE,
114:189-198. Thibodeau SN, Parker AS and Eckel-Passow JE (2013) Ex-
Kirchhoff M, Rose H and Lundsteen C (2001) High resolution perimental designs for array comparative genomic hybrid-
comparative genomic hybridisation in clinical cytogenetics. ization technology. Cytogenet Genome Res 139:250-257.
J Med Genet 38:740-744. McNamara LE, Dalby MJ and Tsimbouri MP (2014) The use of
Langer PR, Waldrop AA and Ward DC (1981) Enzymatic synthe- microarrays and fluorescence in situ hybridization for the
sis of biotin-labeled polynucleotides: Novel nucleic acid af- study of mechanotransduction from topography. Methods
finity probes. Proc Natl Acad Sci USA 78:6633-6637. Cell Biol 119:293-309.
Le Caignec C, Spits C, Sermon K, De Rycke M, Thienpont B, Meltzer PS, Guan XY, Burgess A and Trent JM (1992) Rapid gen-
Debrock S, Staessen C, Moreau Y, Fryns JP, Van eration of region specific probes by chromosome micro-
Steirteghem A, et al. (2006) Single-cell chromosomal im- dissection and their application. Nat Genet 1:24-28.
balances detection by array CGH. Nucleic Acids Res Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR,
34:e68. Carter NP, Church DM, Crolla JA, Eichler EE, Epstein CJ,
Le Scouarnec S and Gribble SM (2012) Characterising chromo- et al. (2010) Consensus statement: Chromosomal micro-
some rearrangements: Recent technical advances in molecu- array is a first-tier clinical diagnostic test for individuals
lar cytogenetics. Heredity (Edinb) 108:75-85. with developmental disabilities or congenital anomalies.
Lee JA, Carvalho CM and Lupski JR (2007) A DNA replication Am J Hum Genet 86:749-764.
mechanism for generating nonrecurrent rearrangements as- Moorhead PS, Nowell PC, Mellman WJ, Battips DM and Hunger-
sociated with genomic disorders. Cell 131:1235-1247. ford DA (1960) Chromosome preparations of leukocytes
Riegel 207

cultured from human peripheral blood. Exp Cell Res Priest JR, Girirajan S, Vu TH, Olson A, Eichler EE and Portman
20:613-616. MA (2012) Rare copy number variants in isolated sporadic
Ness GO, Lybaek H and Houge G (2002) Usefulness of high- and syndromic atrioventricular septal defects. Am J Med
resolution comparative genomic hybridization (CGH) for Genet 158A:1279-1284.
detecting and characterizing constitutional chromosome ab- Rabbani B, Tekin M and Mahdieh N (2014) The promise of
normalities. Am J Med Genet 113:125-136. whole-exome sequencing in medical genetics. J Hum Genet
Niazi M, Coleman DV and Loeffler FE (1981) Trophoblast sam- 59:5-15.
pling in early pregnancy. Culture of rapidly dividing cells Rafati M, Seyyedaboutorabi E, Ghadirzadeh MR, Heshmati Y,
from immature placental villi Br J Obstet Gynaecol Adibi H, Keihanidoust Z, Eshraghian MR, Javadi GR, Das-
88:1081-1085. tan J, Mosavi-Jarrahi A, et al. (2012) “Familial” vs. “Spo-
Nicholl J, Waters W, Mulley JC, Suwalski S, Brown S, Hull Y, radic” intellectual disability: Contribution of common
Barnett C, Haan E, Thompson EM, Liebelt J, et al. (2014) microdeletion and microduplication syndromes. Mol
Cognitive deficit and autism spectrum disorders: Prospec- Cytogenet 5:9.
tive diagnosis by array CGH. Pathology 46:41-45. Reichman J (2000) Handbook of optical filters for fluorescence
Nietzel A, Rocchi M, Starke H, Heller A, Fiedler W, Wlodarska I, microscopy. Chroma Technology, Brattleboro, 36 pp.
Loncarevic IF, Beensen V, Claussen U and Liehr T (2001) A Rickman L, Fiegler H, Carter NP and Bobrow M (2005) Prenatal
new multicolor-FISH approach for the characterization of diagnosis by array-CGH. Eur J Med Genet 48:232-240.
marker chromosomes: Centromere-specific multicolor- Riggs ER, Church DM, Hanson K, Horner VL, Kaminsky EB,
FISH (cenM-FISH). Hum Genet 108:199-204. Kuhn RM, Wain KE, Williams ES, Aradhya S, Kearney
Nowell PC (1960) Phytohemagglutinin: An initiator of mitosis in HM, et al. (2012) Towards an evidence-based process for
cultures of normal human leukocytes. Cancer Res 20:462- the clinical interpretation of copy number variation. Clin
466. Genet 81:403-412.
Painter TS (1923) Studies in mammalian spermatogenesis II. The Rosner M, Pergament E, Andriole S, Gebb J, Dar P and Evans MI
spermatogenesis of man. J Exp Zool 37:291-321. (2013) Detection of genetic abnormalities by using CVS and
Palmer E, Speirs H, Taylor PJ, Mullan G, Turner G, Einfeld S, FISH prior to fetal reduction in sonographically normal ap-
Tonge B and Mowat D (2014) Changing interpretation of pearing fetuses. Prenat Diagn 33:940-944.
chromosomal microarray over time in a community cohort Rouillard JM, Herbert CJ and Zuker M (2002) OligoArray: Ge-
with intellectual disability. Am J Med Genet A 164:377- nome-scale oligonucleotide design for microarrays. Bio-
385. informatics 18:486-487.
Pardue ML and Gall JG (1970) Chromosomal localization of Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM
mouse satellite DNA. Science 168:1356-1358. (1998) Telomere length dynamics in human lymphocyte
Perry GH, Ben-Dor A, Tsalenko A, Sampas N, Rodriguez-Reven- subpopulations measured by flow cytometry. Nat Bio-
ga L, Tran CW, Scheffer A, Steinfeld I, Tsang P, Yamada technol 16:743-747.
NA, et al. (2008) The fine-scale and complex architecture of Sanders SJ, Ercan-Sencicek AG, Hus V, Luo R, Murtha MT,
human copy-number variation. Am J Hum Genet 82:685- Moreno-De-Luca D, Chu SH, Moreau MP, Gupta AR,
695. Thomson SA, et al. (2011) Multiple recurrent de novo
Pinkel D, Gray JW, Trask B, van den Engh G, Fuscoe J and van CNVs, including duplications of the 7q11.23 Williams syn-
Dekken H (1986a) Cytogenetic analysis by in situ hybridiza- drome region, are strongly associated with autism. Neuron
tion with fluorescently labeled nucleic acid probes. Cold 70:863-885.
Spring Harbor Symp Quant Biol 51:151-157. Schinzel A (2001) Catalogue of Unbalanced Chromosome Aber-
Pinkel D, Straume T and Gray JW (1986b) Cytogenetic analysis rations in Man. 2nd edition. Walter de Gruyter, Berlin,
using quantitative, high-sensitivity, fluorescence hybridiza- 966 pp.
tion. Proc Natl Acad Sci USA 83:2934-2938. Schinzel A, Riegel M, Baumer A, Superti-Furga A, Moreira LM,
Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J Santo LD, Schiper PP, Carvalho JH and Giedion A (2013)
and Gray JW (1988) Fluorescence in situ hybridization with Long-term follow-up of four patients with Langer-Giedion
human chromosome-specifi c libraries: Detection of trisomy syndrome: Clinical course and complications. Am J Med
21 and translocations of chromosome 4. Proc Natl Acad Sci Genet A 161:2216-2225.
USA 85:9138-9142. Schou KV, Kirchhoff M, Nygaard U, Jørgensen C and Sundberg
Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Col- K (2009) Increased nuchal translucency with normal karyo-
lins C, Kuo WL, Chen C, Zhai Y, et al. (1998) High resolu- type: A follow-up study of 100 cases supplemented with
tion analysis of DNA copy number variations using compar- CGH and MLPA analyses. Ultrasound Obstet Gynecol
ative genomic hybridization to microarrays. Nat Genet 34:618-622.
20:207-211. Schröck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Fer-
Pita M, Orellana J, Martínez-Rodríguez P, Martínez-Ramírez A, guson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I,
Fernández-Calvín B and Bella JL (2014) FISH methods in Soenksen D, et al. (1996) Multicolor spectral karyotyping of
cytogenetic studies. Methods Mol Biol 1094:109-135. human chromosomes. Science 273:494-497.
Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Perga- Schröck E, Zschieschang P, O’Brien P, Helmrich A, Hardt T,
menschikov A, Williams CF, Jeffrey SS, Botstein D and Matthaei A and Stout-Weider K (2006) Spectral karyo-
Brown PO (1999) Genome-wide analysis of DNA copy- typing of human, mouse, rat and ape chromosomes - Appli-
number changes using cDNA microarrays. Nat Genet cations for genetic diagnostics and research. Cytogenet Ge-
23:41-46. nome Res 114:199-221.
208 Human molecular cytogenetics

Seabright M (1971). A rapid banding technique for human chro- van der Veken LT and Buijs A (2011) Array CGH in human leu-
mosomes. Lancet 2:971-972. kemia: From somatics to genetics. Cytogenet Genome Res
Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P, 135:260-270.
Mànér S, Massa H, Walker M, Chi M, et al. (2004) Large- Vermeesch JR, Fiegler H, de Leeuw N, Szuhai K, Schoumans J,
scale copy number polymorphism in the human genome. Ciccone R, Speleman F, Rauch A, Clayton-Smith J, Van
Science 305:525-528. Ravenswaaij C, et al. (2007) Guidelines for molecular
Shao L, Kang SH, Li J, Hixson P, Taylor J, Yatsenko SA, Shaw karyotyping in constitutional genetic diagnosis. Eur J Hum
CA, Milosavljevic A, Chang CC, Cheung SW, et al. (2010) Genet 15:1105-1114.
Array comparative genomic hybridization detects chromo- Vermeesch JR, Brady PD, Sanlaville D, Kok K and Hastings RJ
somal abnormalities in hematological cancers that are not (2012) Genome-wide arrays: Quality criteria and platforms
detected by conventional cytogenetics. J Mol Diagn to be used in routine diagnostics. Hum Mutat 33:906-915.
12:670-679. Vetro A, Bouman K, Hastings R, McMullan DJ, Vermeesch JR,
Shimizu K, Wakui K, Kosho T, Okamoto N, Mizuno S, Itomi K, Miller K, Sikkema-Raddatz B, Ledbetter DH, Zuffardi O
Hattori S, Nishio K, Samura O, Kobayashi Y, et al. (2013) and van Ravenswaaij-Arts CM (2012) The introduction of
Microarray and FISH-based genotype-phenotype analysis arrays in prenatal diagnosis: A special challenge. Hum
of 22 Japanese patients with Wolf-Hirschhorn syndrome. Mutat 33:923-929.
Am J Med Genet A [Epub ahead of print]. Vissers LE and Stankiewicz P (2012) Microdeletion and micro-
Simons A, Sikkema-Raddatz B, de Leeuw N, Konrad NC, Has- duplication syndromes. Methods Mol Biol. 838:29-75.
tings RJ and Schoumans J (2012) Genome-wide arrays in Vissers LE, de Vries BB, Osoegawa K, Janssen IM, Feuth T,
routine diagnostics of hematological malignancies. Hum Choy CO, Straatman H, van der Vliet W, Huys EH, van Rijk
Mutat Mutat 33:941-948. A, et al. (2003) Array-based comparative genomic hybrid-
Snijders AM, Nowak N, Segraves R, Blackwood S, Brown N, ization for the genomewide detection of submicroscopic
Conroy J, Hamilton G, Hindle AK, Huey B, Kimura K, et al. chromosomal abnormalities. Am J Hum Genet 73:1261-
(2001) Assembly of microarrays for genome-wide measure- 1270.
ment of DNA copy number. Nat Genet 29:263-264. Vorsanova SG, Yurov YB and Iourov IY (2010) Human inter-
phase chromosomes: A review of available molecular
Solinas-Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner
cytogenetic technologies. Mol Cytogenet 3:1-15.
A, Döhner H, Cremer T and Lichter P (1997) Matrixbased
comparative genomic hybridization: Biochips to screen for Vulto-van Silfhout AT, van Ravenswaaij CM, Hehir-Kwa JY,
genomic imbalances. Genes Chromosomes Cancer 20:399- Verwiel ET, Dirks R, van Vooren S, Schinzel A, de Vries
407. BB and de Leeuw N (2013) An update on ECARUCA, the
European Cytogeneticists Association Register of Unbal-
Speicher MR, Ballard SG and Ward DC (1996) Karyotyping hu-
anced Chromosome Aberrations. Eur J Med Genet 56:471-
man chromosomes by combinatorial multi-fl uor FISH. Nat
474.
Genet 12:368-375.
Waldeyer W (1888) Über Karykinese und ihre Beziehung zu den
Steele MW and Breg Jr WR (1966) Chromosome analysis of hu-
Befruchtungsvorgängen. Arch Mikrosk Anat 32:1-112.
man amniotic-fluid cells. Lancet 1:383-385.
Wegner DE (1999) Diagnostic Cytogenetics. Springer, Berlin,
Stefansson H, Meyer-Lindenberg A, Steinberg S, Magnusdottir 460 pp.
B, Morgen K, Arnarsdottir S, Bjornsdottir G, Walters GB, Weise A, Gross M, Mrasek K, Mkrtchyan H, Horsthemke B,
Jonsdottir GA, Doyle OM, et al. (2014) CNVs conferring Jonsrud C, Von Eggeling F, Hinreiner S, Witthuhn V, Claus-
risk of autism or schizophrenia affect cognition in controls. sen U, et al. (2008) Parental-origin-determination fluores-
Nature 505:361-6. cence in situ hybridization distinguishes homologous human
Stumm M, Wegner RD, Bloechle M and Eckel H (2006) Inter- chromosomes on a single-cell level. Int J Mol Med 21:189-
phase M-FISH applications using commercial probes in pre- 200.
natal and PGD diagnostics. Cytogenet Genome Res Weise A, Mrasek K, Klein E, Mulatinho MV, Llerena Jr JC,
114:296-301. Hardekopf D, Pekova S, Bhatt S, Kosyakova N and Liehr T
Sun Z, Liu P, Jia X, Withers MA, Jin L, Lupski JR and Zhang F (2012) Microdeletion and microduplication syndromes. J
(2013) Replicative mechanisms of CNV formation preferen- Histochem Cytochem 60:346-58.
tially occur as intrachromosomal events: Evidence from Wellcome Trust Case Control Consortium, Craddock N, Hurles
Potocki-Lupski duplication syndrome. Hum Mol Genet ME, Cardin N, Pearson RD, Plagnol V, Robson S, Vukcevic
22:749-756. D, Barnes C, Conrad DF, et al. (2010) Genome-wide associ-
Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier ation study of CNVs in 16,000 cases of eight common dis-
H, Heetebrij RJ, Talman EG, Raap AK and Vrolijk J (1999) eases and 3,000 shared controls. Nature 464:713-720.
New strategy for multi-colour fluorescence in situ hybridisa- Wells D and Delhanty JD (2000) Comprehensive chromosomal
tion: COBRA: COmbined Binary RAtio labelling. Eur J analysis of human preimplantation embryos using whole ge-
Hum Genet 7:2-11. nome amplification and single cell comparative genomic hy-
Telenius H, Carter NP, Bebb CE, Nordenskjöld M, Ponder BA bridization. Mol Hum Reprod 6:1055-1062.
and Tunnacliffe A (1992) Degenerate oligonucleotide- Wiszniewska J, Bi W, Shaw C, Stankiewicz P, Kang SH, Pursley
primed PCR: General amplification of target DNA by a sin- AN, Lalani S, Hixson P, Gambin T, Tsai CH, et al. (2014)
gle degenerate primer. Genomics 13:718-725. Combined array CGH plus SNP genome analyses in a single
Tijo JH and Levan A (1956) The chromosome number of man. assay for optimized clinical testing. Eur J Hum Genet
Hereditas 42:1-6. 22:79-87.
Riegel 209

Xing J, Zhang Y, Han K, Salem AH, Sen SK, Huff CD, Zhou Q, Chromosomal Variation in Man Online Database:
Kirkness EF, Levy S, Batzer MA and Jorde LB (2009) Mo- http://www.wiley.com/legacy/products/sub-
ject/life/borgaonkar/access.html (2014-01-28).
bile elements create structural variation: Analysis of a com- Cytogenetic Data Analysis System (CyDAS):
plete human genome. Genome Res 19:1516-1526. http://www.cydas.org/ (2014-01-28).
Database of genomic structural variation (bdVar):
Yunis JJ (1976) High resolution of human chromosomes. Science http://www.ncbi.nlm.nih.gov/dbvar/ (2014-01-28).
191:1268-1270. Ensembl: www.ensembl.org/ (2014-01-28).
European Cytogeneticists Association Register of Unbalanced
Chromosome Aberrations (ECARUCA): www.ecaruc.net
Internet Resources (2014-01-28).
The International Standards for Cytogenomic Arrays (ISCA)
Centre for the Development and Evaluation of Complex Interven- Consortium:https://www.iscaconsortium.org/index.php
(2014-01-28).
tions for Public Health Improvement (DECIPHER) project: Small supernumerary marker chromosomes:
http://decipher.sanger.ac.uk (2014-01-28). http://ssmc-tl.com/sSMC.html (2014-01-28).

The Chromosome Anomaly Collection: License information: This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and
http://www.ngrl.org.uk/wessex/collection/ (2014-01-28). reproduction in any medium, provided the original work is properly cited.