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Molecular cytogenetic techniques for identification of copy-number variations

Eid, Ola M.

Middle East Journal of Medical Genetics: January 2017 - Volume 6 - Issue 1 - p 1–12
doi: 10.1097/01.MXE.0000510784.43101.7a
Review article

In the latter decades, the conventional cytogenetics techniques have experienced significant advancement into molecular cytogenetics technologies. The limitations of conventional cytogenetics techniques have largely been overcome by these new technologies. Molecular cytogenetics techniques are basically used for the identification and delineation of copy-number changes for diagnostic purposes and genetic counseling. This article reviews various molecular cytogenetics methods such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, array-comparative genomic hybridization, and next-generation sequencing techniques.

Department of Human Cytogenetics, National Research Centre, Cairo, Egypt

Correspondence to Ola M. Eid, PhD, Department of Human Cytogenetics, National Research Centre, Cairo 12622, Egypt Tel: ++20 100 177 5606; e-mail:

Received March 31, 2016

Accepted August 10, 2016

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Human cytogenetics is the area of studying of number, configuration, and function of chromosomes relative to the inheritance and origin of pathology (Riegel, 2014). During the latter of the last century, the clinical applications of human cytogenetics have been advanced from the morphological karyotyping techniques using light microscopy into the molecular karyotyping techniques displayed on the computer screen (Mulley and Mefford, 2011).

In 1970s, the conventional karyotyping procedures have been instituted. They were the fundamentals for gene mapping. They allocate genes relative to the visible landmarks along the chromosome. Later on, during 1990s, fluorescence in situ hybridization (FISH) technique has introduced cytogenetics into the molecular era. Since then, FISH technique has significantly upgraded the sensitivity of small chromosomal aberration detection (Haaf, 2000).

Despite the generally used conventional karyotyping techniques being very informative, they are still restricted to the detection of microscopic structural variants, a few megabases in size. On the contrary, molecular karyotyping/cytogenetics approaches can detect the submicroscopic structural variants (Le Scouarnec and Gribble, 2012). The wide gap in resolution between the conventional cytogenetic procedures (megabase pairs) and the molecular procedures (base pair) has been greatly minimized by the introduction of molecular cytogenetic methods (Gouas et al., 2008).

Among the armory of the molecular cytogenetics technologies, there are FISH, multiplex ligation-dependent probe amplification (MLPA), array-comparative genomic hybridization (aCGH), and next-generation sequencing (NGS) techniques (Gouas et al., 2008; Gijsbers and Ruivenkamp, 2011; Le Scouarnec and Gribble, 2012; Riegel, 2014).

Most of the diagnostic and research laboratories are switching from the conventional karyotype techniques to the ‘molecular karyotype’ techniques. Molecular cytogenetics procedures are now deemed the first diagnostic step. This is because of their efficacy for diagnosis of many diseases genetically, such as developmental delay, intellectual disabilities, and congenital abnormalities. Consequently, the applications of the molecular cytogenetics techniques have significantly increased the diagnostic output in the cytogenetics field (Mulley and Mefford, 2011).

The grounds of the molecular karyotyping/cytogenetics techniques depend upon the evaluation of the chromosome content and structure using DNA hybridization instead of the direct monitoring of chromosomes under the microscope. The molecular karyotype techniques can detect the imbalances caused by deletion or duplication of DNA [copy-number variants (CNVs)]. However, with the exception of FISH and NGS techniques, they cannot detect the truly balanced translocations, which is the important variation between the conventional and molecular karyotype techniques. The contemporary advances in molecular karyotyping/cytogenetics techniques allow the researchers to investigate the whole genome for CNVs – in other words, gene dosage alterations at a solitary assay (Mulley and Mefford, 2011).

The aim of this article is to globally review some molecular cytogenetic techniques that are applied for studying chromosomal abnormalities and for evaluation of the copy-number changes.

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Fluorescence in situ hybridization

FISH study was the motivating power in the development of cytogenetic techniques (Riegel, 2014). FISH assay is an extremely powerful and extensively used assay that directly visualizes specific DNA sequence on morphologically preserved cytological specimens such as metaphases, interphase nuclei, and extended chromatin fibers or DNA molecules (Haaf, 2000).

FISH technique was originally developed in late 1960s (John et al., 1969). At that time, radioactive labels were only available. However, enzymatic labeling of nucleic acids with biotin and other haptens offered numerous advantages over radioactive in situ hybridization (Langer et al., 1981). The high spatial resolution, speed, probe stability, and the capability to detect several chromosomal targets in different colors are the major advantages of enzymatic labeling of FISH probe over radioactive labeling (Haaf, 2000).

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Principle of fluorescence in situ hybridization

The basis of FISH study is targeting and denaturing DNA fixed in cells, nuclei, or metaphase chromosomes on the surface of the slide. Next, a complementary single-stranded DNA sequence probe, after its denaturation, would specifically reanneal during the hybridization reaction forming double-stranded DNA (hybrid) molecules. Probe DNA molecules are enzymatically labeled with modified nucleotides. They are hapten-labeled (indirect FISH) and fluorescent-labeled (direct FISH) DNA molecules. After removal of unbound single-stranded DNA and nonspecifically bound DNA from the slide by posthybridization washing, an antifade solution containing 4′,6-diamidino-2 phenylindole is applied to the slide. FISH signals are observed using epifluorescence microscopes with specific filters for identifying fluorochromes. A charge-coupled device camera captures the image, and the fluorescent signals are subsequently quantified (Haaf, 2000; Riegel, 2014) (Fig. 1).

Figure 1

Figure 1

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Types of fluorescence in situ hybridization probes

The selection of the probe is a critical step in FISH study and relies on the application for which FISH is going to be used (Hames and Higgins, 1995). At present, a variety of commercial probes (e.g. whole-chromosome painting probes (WCP), chromosome-arm painting probes, and repetitive centromeric, subtelomeric, telomeric, and locus-specific probes) are available for the identification of particular constitutional and acquired chromosomal anomalies (Riegel, 2014) (Fig. 2). RNA probes are also used in FISH study, and their major applications are to reveal mRNA expressions in cell and tissue material (Olivier et al., 1996).

Figure 2

Figure 2

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Fluorescence in situ hybridization-based technology

The standard FISH has its limitation. It could not identify all of the chromosomes in the whole genome concurrently (Riegel, 2014). Since launching of standard FISH (Pinkel et al., 1986a, 1986b), more sensitive FISH-based techniques have been steadily developed, and several digital imaging systems have been presented for FISH image acquisition, image preprocessing, and digital image analysis. There are various techniques using FISH-based methods for diverse applications, including fiber-FISH, multicolor FISH (M-FISH), spectral karyotyping FISH, and combined binary ratio labeling-FISH (Riegel, 2014). FISH technique can also be used to interphase cells, interphase FISH, which grants the visualization of DNA FISH probes in interphase nuclei (Vorsanova et al., 2010).

Combined binary ratio labeling-FISH, M-FISH, and spectral karyotyping FISH are the most sophisticated FISH-based methods. These methods allow visualization and detection of all chromosomes concurrently through color karyotyping. WCP are used to simultaneously stain each of the 24 human chromosomes with a different color. Several fluorescence dyes are used to label the WCP probes. The required 24 color combinations are achieved through ratio labeling. The resulting chromosome karyotyping is done automatically using commercial software (Schröck et al., 2006; Riegel, 2014).

Collectively, FISH study is a simple technique of wide applications. Although FISH technique is used mainly for studying human genome, it has been effectively used in the study of various genomes (Haaf, 2000).

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Some applications of fluorescence in situ hybridization

Despite the continuous upgrading of conventional cytogenetic techniques, subtle structural rearrangements of chromosomes of less than one band (1–10 MB) remain undetected (as in microdeletion, microduplication syndromes, and cryptic translocations). However, this problem has been resolved with the establishment of FISH technique with its high sensitivity, up to 10 kbp (Sukarova-Angelovska et al., 2007). Using subtelomeric and locus-specific FISH probes in patients having intellectual disability (ID) allows detection of subtle structural abnormalities, which is important from the diagnostic and genetic counseling point of view (Baroncini et al., 2005; Sukarova-Angelovska et al., 2007; Tos et al., 2013).

The existence of de novo marker or derivative chromosomes is quite problematic for genetic counseling, especially in prenatal diagnosis. This is because the identification of marker and derivative chromosomes by conventional cytogenetic techniques is nearly impossible. With the development of M-FISH, identification of marker and derivative chromosomes in a solitary hybridization can be done (Cetin et al., 2005; Wu et al., 2013).

Chromosomal mosaicism is the coexistence of more than one karyotypically cell lines in the same individual. The use of interphase FISH on tissues from different germ layers (mesodermal tissue such as lymphocytes and ectodermal tissue such as buccal epithelial cells) upgrades the precision of detection of hidden mosaicism, especially when mosaic aneuploidy with low-level frequency is doubtful (Nazarenko et al., 1999). Maciel-Guerra et al. (2012) achieved the concluding diagnosis of three patients by FISH by demonstrating hidden mosaicism, confirming the significance of FISH in detection of hidden mosaicism.

FISH on uncultured amniocytes using locus-specific FISH probes offers the chance for fast aneuploidy screening, and around 80–95% of all chromosomal disorders can be discovered within 24 h (Lev et al., 2005). That was confirmed by Liehr and Ziegler (2005), based on their experience of 1200 samples. However, they stated that the test should be used exclusively as a preamble to full chromosome analysis by microscopy. In 2010, Liu et al. (2010) insisted that FISH is a reliable and rapid prenatal diagnostic tool as an adjunct to classical cytogenetic study, and it can be used for rapid and accurate prenatal diagnosis of women with high-risk of maternal serum screening.

Fusion genes are the results of chromosomal translocations and are valuable markers in classification of malignancies and in delineation of risk patients who need different treatment, as in precursor-B cell acute lymphoblastic leukemia, acute myeloid leukemia (Van Dongen et al., 2005), chronic myeloid leukemia (Manaflouyan Khajehmarjany et al., 2015), and renal cell carcinoma (Chen et al., 2015). There are two types of FISH probe design for the fusion genes: fusion-signal FISH probe and split-signal FISH probe. The classical fusion-signal FISH type uses two distinctively labeled probes, green and red, which flank the breakpoint regions of the two genes that are implicated in the translocation. Normally, that is, without chromosomal translocation, two red signals and two green signals are present. In case of a translocation, a red and a green signal will be juxtaposed, presenting a colocalized green/red signal, which will usually appear as a yellow signal, in addition to separate green and red signals of the unaffected chromosomes (Van Dongen et al., 2005; Manaflouyan Khajehmarjany et al., 2015). The split-signal FISH type also uses two distinctively labeled probes that are positioned in only one of the two involved genes, the target gene. They are located at opposite sides of the breakpoint region of the target. Normally, the yellowish coloration of the colocalized green/red signals is present. The translocation will lead to a split of one of the colocalized signals, giving a separate green and red signal in addition to the fused signal of the unaffected chromosome (Van Dongen et al., 2005; Chen et al., 2015).

FISH is a well-established molecular cytogenetic technique, which is regularly used in diagnostic pathology to detect the presence of specific loci in cancer cells (Das and Tan, 2013) – for example, the multitarget UroVysion (Abbott Molecular Inc., USA) FISH probe, which is an eminent test that upgraded cancer diagnosis in urinary cytopathology (Bubendorf and Piaton, 2012). Moreover, PathVysion (Abbott Molecular Inc., USA) HER-2 FISH probe is an assay to detect amplification of the HER2 gene in tissue of breast cancer to determine and select patients suitable for treatment with Trastuzumab. In addition, Vysis ALK Break Apart FISH probe is used to identify rearrangements involving the ALK gene non-small-cell lung cancer tissue to recognize patients appropriate for treatment with crizotinib (Hiraoka, 2014).

FISH is used for chromosomal breakpoint identification. The probes used are mapped to the bands of the cytogenetically assigned breakpoints and selected from the Ensemble Human Genome Browser. The gene content of the breakpoint regions is defined with the Ensemble and the UCSC genome browsers. The probes of large genomic DNA sequences are cloned into cosmids, bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes, or yeast artificial chromosomes. Next, the probes are labeled with Spectrum Orange or Green – dUTP by nick translation. For breakpoints affecting regions harboring genes, additional precise mapping should be undertaken by FISH with fosmid clones (Bertrand et al., 2007; Baptista et al., 2008; De Braekeleer et al., 2011).

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Multiplex ligation-dependent probe amplification

MLPA was initially designated in 2002 by Schouten et al. (2002). It is a multiplex PCR technique that detect abnormal copy numbers of up to 50 different genomic DNA or RNA sequences distinguishing sequences differ in only one nucleotide. A capillary electrophoresis equipment and thermocycler are the only requirements. Up to 96 samples can be processed simultaneously, with the availability of the results within 24 h. The application of MLPA clinically considerably raises the detection rate of various genetic disorders (Stuppia et al., 2012).

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Principle of multiplex ligation-dependent probe amplification technique

The MLPA technique is performed in the following steps: (a) DNA denaturation and hybridization of MLPA probes: the DNA is denatured and incubated overnight with a mixture of MLPA probes – MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences, and the two probe oligonucleotides hybridize immediately to adjacent target sequences; (b) Ligation reaction: only when the two probe oligonucleotides are both hybridized to their adjacent targets can they be ligated during the ligation reaction; (c) PCR: only ligated probes will be exponentially amplified during the subsequent PCR reaction using a single universal dye-labeled primer pair; (d) Separation of amplification products by electrophoresis: the amplification products are separated using capillary electrophoresis; and (e) Data analysis: where aberrant copy numbers are detected by comparing the achieved peak pattern of the sample with that of reference samples. In MLPA, only the ligated products will be amplified, and thus it is not necessary to remove the nonligated probe (a single-tube assay). This makes MLPA easy to perform and especially attractive for application in clinical diagnosis (Gouas et al., 2008; Stuppia et al., 2012) (Fig. 3).

Figure 3

Figure 3

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Probe design and quantification

MLPA probes are composed of two single-stranded DNA half-probes. The two MLPA half-probes should be unique, have a similar GC content, and create products that differ in length. Each probe in an MLPA probe mix has a unique amplicon length, typically ranging from 130 to 500 nucleotide. All ligated probes have identical sequences at their 5′ and 3′ ends, permitting simultaneous amplification in a PCR containing only one (universal) primer. Discrimination of each individual probe after amplification is accomplished by granting all probes a different length, enabling their separation and scoring after capillary electrophoresis. The classical MLPA protocol uses one synthetic and one M13-cloned half-probe. The synthetic left-hand probes, 5’ half-probe (the short half), contain a 19-nucleotide common tail sequence at the 5′ end and a 21–30-nucleotide target-specific sequence at the 3′ end. The M13-cloned right-hand probes, 3′ half-probe (the long half), contain a common tail sequence at the 3′ end and 25–43-nucleotide target-specific sequences M13-cloned at the 5′ end, which is phosphorylated to enable ligation to its sister 5′ half-probe and contain a different so-called nonhybridizing stuffer fragment (of 19–370 nucleotide in between), which is used to produce length differences between the individual probes, to generate probes varying in length from 120 to 480 bp. The length differences between the consecutive amplified products are of 6–9 bp (Sellner and Taylor, 2004; Kozlowski et al., 2008; Stuppia et al., 2012).

The use of two-color labeling on two distinct pairs of universal primers enables a simultaneous capillary electrophoresis analysis of two sets of MLPA products for which size and migration overlap. This tactic increases the multiplexing capacity by a factor of 2, although each set of probes must include its own control probes, and results from the two different MLPA probe sets pooled together must be analyzed separately (White et al., 2004).

MLPA probe mixes contain several internal controls to facilitate subsequent data analysis. Therefore, for screening of possible deletions/duplications in a particular gene, it is essential that the internal controls probes are from unrelated genomic regions, preferably other chromosomes, and from regions in the genome where deletions/duplications are identified to be incompatible with life or to have very distinguished phenotypic consequences, or from regions known to be implicated in other diseases. In addition, MLPA probe mixes contain reference probes that detect sequences that are assumed to have the same copy number in both patient and reference samples and are not (known to be) involved in the disease studied. Furthermore, up to 10 different control fragments are present in MLPA probe mixes; they indicate insufficient sample DNA (Q-fragments), incomplete sample denaturation (D-fragments), and sample switching errors (chromosome X and Y-specific fragments). On the basis of a diploid genome, a 50% signal reduction means deletion (from two copies to one copy), whereas a 50% signal increase means duplication (from two to three copies) (den Dunnen and White, 2006).

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Advantages of multiplex ligation-dependent probe amplification

The MLPA technique has several advantages over other assays. Methods that detect point mutations, such as sequencing, are generally unable to detect copy-number changes. In spite of the fact that Southern blot analysis can detect many aberrations, it will not always detect small deletions and is not ideal as a routine assay. However, quantitative PCR is rapid, potent, and able to detect very small rearrangements but can only analyze one target region per assay. Moreover, breakpoint PCR is a powerful and dependable technique to screen for specific CNVs, but it can be used only when the breakpoints for that specific breakpoint have been identified down to the nucleotide level. Compared with FISH, MLPA not only has the advantage of being a multiplex technique (the workload involved in FISH is significant and not amenable to automation), but also one in which very small (50–70 nucleotide) sequences are targeted, enabling MLPA to identify the frequent, single-gene aberrations, which are too small to be detected by FISH. Furthermore, duplications can be only detected when the duplicated region is rather large; otherwise, the two FISH signals cannot be distinguished. Comparing MLPA with aCGH, MLPA is an economic, technically uncomplicated assay. Although MLPA is not appropriate for genome-wide research screening, it is a worthy substitute to array-based techniques for several routine applications (Sellner and Taylor, 2004; Gouas et al., 2008; Stuppia et al., 2012).

MLPA cannot detect most inversions, balanced translocations, or copy-number changes that lie outside the sequence detected by an MLPA probe. Furthermore, MLPA assay is not able to detect small (point) mutations (den Dunnen and White, 2006; Kozlowski et al., 2008; Stuppia et al., 2012).

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Multiplex ligation-dependent probe amplification variations

Reverse transcriptase MLPA (RT-MLPA) is carried out for mRNA profiling. The RT-MLPA technique begins with the reverse transcription of mRNA into cDNA, because the ligase enzyme cannot ligate probes that are bound to RNA. After this, RT-MLPA continues as a classical MLPA reaction. RT-MLPA is more competent than single-gene analyses by quantitative PCR or Northern blotting, but it is considerably less comprehensive than microarray-based expression profiling. The advantages of RT-MLPA are speed (1–2 days), low RNA requirement, and low cost (Kozlowski et al., 2008).

Although methylation-specific MLPA (MS-MLPA) is carried out for both copy-number quantification and methylation profiling, MS-MLPA is a very valuable technique to diagnose imprinting diseases and to investigate methylation aberrations in tumor samples (Kozlowski et al., 2008; Stuppia et al., 2012).

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Some applications of multiplex ligation-dependent probe amplification

With the advancement of molecular cytogenetic techniques, many microdeletion/microduplication syndromes with developmental delay (DD)/ID are now outlined by MLPA technique. It was stated that ‘using a set of MLPA kits to identify chromosomal imbalances in patients with multiple congenital anomalies and ID is a valuable choice for developing countries’ (Jehee et al., 2011). Moreover, MLPA was suggested as the first screening assay for children suffering from ID with normal karyotypes (Loghmani Khouzani et al., 2014). This was confirmed by Boggula et al., 2014, who concluded that the use of MLPA probes gives good diagnostic yield in patients having DD/ID. In addition, although MLPA cannot investigate the whole genome like cytogenetic microarray, it is still a significant tool to evaluate DD/ID patients because of its easiness and economic costs (Fig. 4).

Figure 4

Figure 4

Slater et al. (2003) stated that MLPA is a rapid, flexible, sensitive, and robust test for prenatal aneuploidy detection. They conducted a blind, prospective trial using MLPA prenatal detection of common aneuploidies (13, 18, 21, X, and Y) on amniotic samples referred for routine analysis. Sex determination was also 100% accurate, which was confirmed by Hamidah et al., 2014. In addition, MLPA provides an accurate method in the prenatal diagnosis of high-risk pregnant women from Duchenne muscular dystrophy families (Li et al., 2013). Moreover, MLPA is an effective complement for α-thalassaemia gene deletion detection. Combination of gap-PCR with MLPA for α-thalassaemia gene deletion detection can prevent the missing of gene deletion, and false-positive or false-negative misdiagnosis of α-thalassemia in prenatal diagnosis (Chen et al., 2013).

Moreover, the standard methods used for methylation study can detect complete absence of either methylated or nonmethylated alleles but generally fail to detect alterations in the proportion of methylated and unmethylated alleles. Dikow et al., 2007; Li et al., 2008; Priolo et al., 2008, concluded that MS-MLPA is an accurate, sensitive, reliable method to detect changes in both CpG methylation and copy number in one simple reaction, for genetic diagnosis of imprinting genetic diseases such as Prader–Willi/Angelman syndromes and Beckwith–Wiedemann/Silver–Russell syndrome.

Al Zaabi et al. (2010); Véronèse et al. (2013); Alhourani et al. (2014) and Konialis et al. (2014), concluded that MLPA is a reliable approach that is cost-effective and can be used as a first-line screen and complementary test to karyotype/FISH analysis, but not an alternative approach for FISH testing of genomic aberration in hematological malignancies such as chronic lymphocytic leukemia.

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Array-comparative genomic hybridization

aCGH is a technology that has the capability to investigate the whole genome for copy-number changes caused by deletions, duplications, aneuploidy, or unbalanced translocations in a single assay, down to as small as that of a single exon on some platforms (Fruhman and van den Veyver, 2010). The early experiments of aCGH (Solinas-Toldo et al., 1997; Pinkel et al., 1998) were carried out to improve the resolution obtained with conventional CGH, which is carried out on metaphase spreads (Kallioniemi et al., 1992). The basic principle of aCGH is the same as that in conventional CGH, where the process involves comparative genomic hybridization using an array rather than a metaphase spread as the substrate (Shinawi and Cheung, 2008; Riegel, 2014).

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Principle of array-comparative genomic hybridization

In aCGH, equal amounts of DNA from a patient and a reference sample fluorescently labeled in different colors are cohybridized to an array containing the DNA targets. The reference DNA is of the same sex of the patient sample DNA. The DNA of the patient and reference are labeled differently with cyanine 3 (Cy3) and cyanine 5 (Cy5). After a series of washing steps, the array is scanned using a microarray scanner, and the intensity of each label is detected and interpreted using specialized software. The subsequent ratio of the fluorescence intensities is proportionate to the ratio of the copy numbers of DNA sequences in the patient and reference genomes. When the intensities of the fluorescent dyes are equal on one probeat-specific genomic region, this means that the patient has equal quantity of DNA in the patient and reference samples at this region. An altered Cy3 : Cy5 ratio indicates a loss or a gain of the patient DNA at that specific genomic region. Mosaicism may have variable detection rates depending on the platforms used (Shinawi and Cheung, 2008; Fruhman and van den Veyver, 2010; Le Scouarnec and Gribble, 2012) (Fig. 5).

Figure 5

Figure 5

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Array-comparative genomic hybridization platform probes

The targeted probes are portions of human genomic DNA in the form of BACs or P1 derived artificial chromosomesclones (size of 75–200 kbp), smaller insert clones such as cosmids (size of 30–40 kbp) and fosmids (size of 40–50 kbp), or oligonucleotides (25–85 mers). The spacing and length of the DNA probes define the genomic resolution of the different aCGH platforms (Shinawi and Cheung, 2008). However, the BACs, which are usually 100–200 kbp, may miss alterations of smaller size but less probably detect alterations of unclear clinical significance. Oligonucleotides, which are much smaller probes, usually 25–60 bp, may detect small alterations that would not be seen using a BAC microarray. However, oligonucleotide arrays more probably detect small alterations of unclear clinical significance (De Paz et al., 2015).

Most of the available aCGH platforms are targeted microarrays. They are intended to detect aneuploidies, fully described microdeletion/microduplication syndromes, as well as subtelomeric and other unbalanced chromosomal rearrangements. However, in whole-genome aCGH platforms, the targets are equally spaced with coverage of approximately one probe per 6 kbp to one probe per 70 kbp. Moreover,whole-genome oligonucleotide arrays have the ability to examine changes smaller than the average BAC size with higher resolution and enhanced dynamic range (signal to noise ratio) (Shinawi and Cheung, 2008).

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Advantages and limitations of array-comparative genomic hybridization

aCGH can examine hundreds or thousands of loci in a single assay. Moreover, it can detect any DNA dosage imbalance including aneuploidies, deletions, and duplications with a higher resolution than conventional cytogenetic and conventional CGH techniques (Gouas et al., 2008). However, aCGH cannot recognize the structural rearrangements of abnormal chromosomes. In addition, it cannot identify the balanced rearrangements such as translocations and inversions. Furthermore, aCGH fails to detect polyploidies, and low-grade mosaicism may be detected with difficulty. However, interpretation difficulties are the major limitations of aCGH. The majority of the false-negative results are commonly because of poor hybridization or imperfect coverage of the genomic region of interest. Interpretation difficulties are also due to the presence of CNVs with unclear clinical significance in the human genome (Gouas et al., 2008). CNVs are frequently found, depending on the resolution and representation of the genome of the microarray; for example, Friedman et al., 2006, reported that 11 de novo copy-number changes were found in 100 children with ID using a high-density oligonucleotide array, which were considered to be clinically significant. However, on average, 30 CNVs were detected in every child of this study.

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Some applications of array-comparative genomic hybridization

aCGH can detect various number of DNA copies scattered throughout the human genome. Some of these aberrations are apparently benign CNVs and are usually inherited from a parent (Lee et al., 2007). Alterations present in one of the healthy parents or in independent normal controls are of no direct phenotypic consequences. However, the genetic counseling may be difficult because of the variable expressivity of the phenotype. The publicly available CNV databases help decision-making about the clinical significance of the imbalances detected by microarrays, such as Database of Genomic Variants ( It might be necessary to investigate the parents and other family members to interpret and explain these results. However, de novo copy-number imbalances are considered pathogenic. This is confirmed if the affected area contains gene(s) with functions in agreement with the abnormal clinical findings or formerly described in patients having similar genomic imbalance and similar phenotype (Shinawi and Cheung, 2008). Therefore, the interpretation of the clinical significance of CNV of the aCGH is a complex process, and professional guidelines should be followed (Kearney et al., 2011). Common policies have been proposed to help interpret CNV findings, but no general criteria have been established so far. CNVs are classified into different categories: benign CNV (normal genomic variant); benign CNV; CNV with uncertain clinical relevance or variants of uncertain significance; and CNV with potential clinical relevance or pathogenic variants (Riegel, 2014).

High-resolution aCGH is used to determine the breakpoints of genomic imbalances in known microdeletion/duplication syndromes. Consequently, correlation of different elements of the phenotype with the genes within the imbalanced genomic region and creation of a deletion chart is carried out. Usually, it is predicted that the severity of the phenotype of microdeletion/duplication syndromes is correlated to the extent of the deletions (Ben-Shachar et al., 2008; Maas et al., 2008). Moreover, aCGH designed for specific chromosomes is used to identify small deletions and accurately map the breakpoints of genomic imbalances of specific syndromes (Johnston et al., 2007).

Clinically, aCGH can be used to increase the diagnostic yield in patients with IDs (Schoumans et al., 2005; Rosenberg et al., 2006; Engels et al., 2007). The usefulness of aCGH in the detection of pathogenic CNVs in patients with idiopathic neurodevelopmental disorders was confirmed by Bartnik et al., 2014. Moreover, application of aCGH on a large scale upgrades the detection yield in patients with Autism Spectrum Disorders and paves the way for the identification of new autism genes (Shinawi and Cheung, 2008).

For prenatal screening, Le Caignec et al. (2005) demonstrated the higher sensitivity of aCGH over conventional cytogenetic methods. Therefore, aCGH has the efficiency to substitute conventional cytogenetics in most of the prenatal diagnoses (Lapaire et al., 2007).

In cancer, somatic chromosomal dosage alterations and rearrangements often take place and contribute to its pathogenesis. The use of aCGH to detect these aberrations offers data on important cancer genes that can be used in diagnosis and tumor classification (Jong et al., 2007) and in prediction of tumor progression and prognosis (Blaveri et al., 2005; Lai et al., 2007). However, the application of aCGH for prognosis is relatively limited. This is because of its inability to detect balanced translocations resulting in fused genes, the frequent pathogenesis of cancer (Mitelman et al., 2007). Nevertheless, for example, chronic lymphocytic leukemia is distinctive among leukemia, as copy-number changes are generally seen and are associated with prognostic significance (Döhner et al., 2000). Patel et al. (2008), using a custom-designed aCGH for chronic lymphocytic leukemia, demonstrated the robustness, high sensitivity, and high specificity of this technique.

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Next-generation sequencing

Sequencing the human genome by conventional Sanger sequencing required over a decade of universal effort (Lander et al., 2001; Venter et al., 2001). In 2005, the evolution of NGS technologies permitted sequencing of a whole human genome to be done in a few days and with less expenses. NGS allows sequencing of millions of DNA molecules concurrently after library preparation of fragments, to generate sequence reads. Sequence reads are aligned to the reference genome and base variants to detect small insertions/deletions (indels) and structural variants (SVs) of 450 bp (Le Scouarnec and Gribble, 2012). Therefore, it can be applied to detect multiple sequence variations (single and multinucleotide variants, insertions, deletions, and gene CNVs from DNA). It can also be used to analyze gene expression levels by quantitatively measuring the levels of mRNA, microRNA, and factors affecting them, such as gene promoter methylation (Ballester et al., 2016).

NGS has the ability to produce an enormous volume of data cheaply, up to one billion short reads per instrument run. The major use of NGS is the resequencing of human genomes, reinforcing our comprehension of the effect of the genetic differences on health and disease (Metzker, 2010). Moreover, it has paved the way for enormous variety of applications such as extensive gene expression studies and whole-genome sequencing of many organisms (Le Scouarnec and Gribble, 2012).

Optical imaging-based NGS technologies using sensitive optical imaging is the most popular NGS technology used by commercially available platforms (Ballester et al., 2016). The most commonly used NGS platforms at present are industrialized by Illumina (Genome Analyzer/MiSeq, HiSeq, NextSeq, USA), Roche (454 Life Sciences, USA) and Applied Biosystems/Life Technologies (SOLiD, USA) (Le Scouarnec and Gribble, 2012; Ballester et al., 2016). However, owing to relatively long work flow and higher cost of sequencing of the 454 Life Sciences sequencer and to the limited read lengths of the SOLiD sequencer, Illumina sequencers are the most widely used NGS platforms. Moreover, a revolutionary nonoptical sequencing NGS technology was developed using a semiconductor chip (Ion Torrent Technology, USA), which makes sequencers using this technology relatively compact and robust (Ballester et al., 2016). Nevertheless, the technology of NGS is still under advancement. Although the read lengths currently range from 30 to 400 bp depending on the platform, third-generation platforms could produce reads reaching up to a few kilobases (Metzker, 2010).

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Principle of next-generation sequencing

Principally, NGS technologies include a number of steps that are grouped generally as template preparation, sequencing and imaging, and data analysis. There are two methods used in preparing templates for NGS reactions: clonally amplified templates originating from single DNA molecules, and single DNA molecule templates. The sequencing step could be carried out by cyclic reversible termination, single-nucleotide addition, and real-time sequencing. Sequencing by ligation is an approach in which DNA polymerase is replaced by DNA ligase. Imaging step is coupled with these sequencing strategies and ranged from measuring bioluminescent signals to four-color imaging of single molecular events. The capacious data produced by these NGS platforms place substantial demands on information technology in terms of data storage, tracking, and quality control (Metzker, 2010) (Fig. 6).

Figure 6

Figure 6

Information obtained by read mapping and sequence coverage allows the detection of SVs, making NGS an appealing substitute to array-based assays. By using the high-throughput NGS, all types and sizes of SVs can supposedly be identified, breakpoints can be determined with high resolution, down to the base pair level, and complex rearrangements can be deciphered with the prospect to investigate multiple breakpoints in a single experiment (Le Scouarnec and Gribble, 2012).

Four different approaches have been designated to characterize SVs: (i) read-depth analysis; (ii) read-pair analysis; (iii) split-read analysis; and (iv) assembly methods, all of which can theoretically identify all types of rearrangements including copy neutral rearrangements (inversions and translocations) (Le Scouarnec and Gribble, 2012). On the basis of one or more of these methods, a diversity of tools have been evolved to analyze chromosomal rearrangements according to the genomic regions affected, the size range, and breakpoint precision (Medvedev et al., 2009; Alkan et al., 2011; Mills et al., 2011).

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Some of next-generation sequencing applications

The production of large numbers of low-cost reads makes the NGS platforms useful for many applications. These include variant discovery by resequencing targeted regions of interest or whole genomes, cataloguing the transcriptomes of cells and tissues (RNA-seq), and genome-wide profiling of epigenetic marks and chromatin structure using other seq-based methods (Metzker, 2010).

Human genome studies aim to catalog single-nucleotide variants and SVs and their association with phenotypic differences, with the eventual goal of personalized genomics for medical purposes. Applying NGS to whole human genomes by different groups of scientists and comparing the results with the reference genome, fewer SVs were found. This is important because undiscovered SVs could account for a substantial fraction of the total number of sequence variants, many of which could be potentially causative in disease (Metzker, 2010).

NGS has also updated the perception of cancer genomes by determining not only the full spectrum of somatic point mutations (Mardis et al., 2009; Pleasance et al., 2010) but also granting more intuitions into complex whole-genome acquired rearrangements (Campbell et al., 2008; Stephens et al., 2009). These researches demonstrated that intrachromosomal and interchromosomal somatic rearrangements can be identified, not imagined. This is partly because they involve small aberrations beyond the resolution of prior molecular cytogenetics methods. This confirms the efficacy of NGS in studying rearrangements (Meyerson et al., 2010). Therefore, the discovery of fusion genes produced from these rearrangements and its potential functional consequences is significantly facilitated. Moreover, transcriptome sequencing using next-generation technologies can detect or validate presumed fusion transcripts in a high-throughput manner (Maher et al., 2009). Advancements in sequencing technology enabled investigators to study cancer genomes in greater breadth and depth; the field has produced novel insights into tumor pathogenesis, identified clinically useful biomarkers, and developed increasingly precise diagnostics and targeted therapeutics (Shen et al., 2015).

Finally, despite all the enthusiasm about NGS and its possibilities, it has its own limitations, including the problem of having too many variants, the problem of limited coverage, sequencing depth, and accuracy, as well as the problem of quality control (Lohmann and Klein, 2014).

Moreover, despite the vast prospect of NGS, array technology has developed lately and is still suitable for a wide variety of research projects. On top of robustness, flexibility, and low input material required, array technologies do not demand as several resources as NGS technologies in the form of tools and computational power. Arrays also provide the opportunity to study a considerable number of samples in a cost-effective manner (Le Scouarnec and Gribble, 2012).

Array-based methods have discovered an unpredicted level of rearrangement complexity such as imbalances in apparent balanced translocations (Gribble et al., 2005; Howarth et al., 2011), but they are mostly limited to the detection of CNVs, and FISH is still essential to distinguish tandem from dispersed duplications and decipher complex rearrangements (Le Scouarnec and Gribble, 2012).

However, third-generation sequencing technologies (Metzker, 2010) will offer longer reads more economically, and will help overcome these issues. Up until now, NGS technologies have broadly been used to study the human genome. Moreover, complete sequencing of more than a thousand organisms has now been completed and hundreds more are in progress (Le Scouarnec and Gribble, 2012).

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Summary and conclusion

The evolution and progress of the diverse molecular cytogenetics protocols are the result of the efforts of many diagnostic and research scientists from different research groups worldwide. These techniques have been constantly upgraded, and it is difficult to cover every aspect and modification in the field of molecular cytogenetics.

The techniques reviewed here offer information on the human genome at various levels of resolution and have potency for diagnostic and research purposes (Table 1). The resolution for studying chromosomes has improved from few megabases to a single nucleotide. Molecular cytogenetics technologies expedite higher resolutions through genome-wide screening for submicroscopic genomic CNVs.

Table 1

Table 1

In clinical diagnosis, molecular cytogenetics techniques are powerful genomic technologies to evaluate diverse disorders. Currently, the challenge to provide the best approach to detect the genetic causes of diseases is increasing. It has become more difficult to precisely deduce which syndrome affects an individual based only on the clinical examination, as the number of recognized genetic syndromes and chromosomal abnormalities has increased and as the clinical characteristics of those syndromes overlap. At present, the detection of large numbers of CNVs using molecular cytogenetic approaches in patients and healthy individuals has been considered a diagnostic drawback because of interpretation difficulties. In general, imbalances that are cytogenetically visible (several Mbp in size) lead to severe clinical phenotypes of specific syndromes or clinical features. However, the improved resolution of chromosome analysis has increased the number of the benign genomic changes. Therefore, the presence of variants of unknown clinical significance will considerably increase. Nevertheless, the main aim of cytogenetics will remain the study of the genomic organization, as well as the structure, function, and evolution of chromosomes, regardless of the development of different molecular cytogenetics techniques that identify chromosomal imbalances and CNVs in the human genome.

Advanced research into the delineation of human genomic changes, as well as collaborations between clinicians, researchers, and bioinformaticians, are required to develop more thorough human genetic variation maps and to enable more precise interpretations of the clinical impact of genomic imbalances.

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Conflicts of interest

There are no conflicts of interest.

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array-comparative genomic hybridization and next-generation sequencing techniques; fluorescence in situ hybridization; multiplex ligation-dependent probe amplification

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