Human interphase chromosomes: a review of available molecular cytogenetic technologies

Human karyotype is usually studied by classical cytogenetic (banding) techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB). Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions) being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations). Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB) allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.


Introduction
Currently, it is estimated that no fewer than 1 million cytogenetic and molecular cytogenetic analyses are performed per year representing the standard of care in several fields of medicine and the routine clinical workup for numerous patients suffering from congenital malformations, mental diseases, cancers, or reproductive problems [1]. Molecular cytogenetic techniques have been repeatedly proven effective in diagnostics and have been recognized as a valuable addition or even alternative to chromosomal banding [2][3][4]. Furthermore, contemporary basic biomedical research widely applies molecular cytogenetic technologies [5][6][7]. Browsing the most popular scientific resources would undoubtedly return several tens of thousands of articles, which mention at least one molecular cytogenetic technique (for more details refer to [3] and web page about multicolor fluorescence in situ hybridization at http://www.med. uni-jena.de/fish/mFISH/mFISHlit.htm managed by Dr. Thomas Liehr, Jena, Germany). Thus, one can be certain that it is hard to overestimate the role of molecular cytogenetics in current biomedicine.
Looking through the voluminous amount of reviews dedicated to molecular cytogenetics, we have found occasional descriptions of both technological and theoretical side of visualizing human chromosomes in interphase. Consequently, we were forced to conclude that undeservedly little attention is paid to interphase molecular cytogenetics in modern biomedical literature. Additionally, technical side of the application is even more rarely addressed. To fill this gap, we have attempted to give an overview of currently applied molecular cytogenetic techniques with a special emphasis on their technological abilities for studying human interphase chromosomes.

Molecular cytogenetic techniques, their resolution and potential for single-cell analysis of interphase chromosomes
The overwhelming majority of molecular cytogenetic techniques are based on hybridization. There are currently two essential platforms available for developments in molecular cytogenetics: fluorescence in situ hybridization (FISH) including comparative genomic hybridization (CGH) [3,36] and peptide nucleic acid (PNA) probing for analysis of chromosomal DNA [37,38]. Alternatively, another technique uses primed in situ labelling (PRINS) reaction [37,38]. The resolution and level of excellence of all these techniques are established against cytogenetic banding analysis, which remains the golden standard in this instance [36].Single-cell molecular cytogenetic analysis can be performed either through analysis of metaphase plates or through analysis of interphase nuclei. Studying metaphase plates has been long described to be successful using several detection technologies (i.e. spectral karyotyping -SKY or multicolor FISH -MFISH) and different DNA probe sets (chromosome-enumeration/centromeric, site-specific, wholepainting, microdissected) [2,3,[5][6][7][9][10][11][12][13]30,36,[39][40][41][42][43][44][45][46]. In general, if modified, almost all these techniques can be applied to interphase cells, but this "transfer of technology" requires significant efforts [2,3,7,10,12,13,[30][31][32][33]35,47]. Generally, all molecular cytogenetic assays that provide for visualization of genomic loci in an interphase nucleus are termed interphase FISH or I-FISH [35]. Table 1 gives an overview of molecular cytogenetic techniques that are used for metaphase and interphase analysis with special attention to the resolution and to the modifications for studying single cells. The impossibility of listing all known molecular cytogenetic approaches seems to be apparent, but even a short description of such techniques (Table 1) shows molecular cytogenetics able to perform high-resolution analysis of chromosomal structure and behavior at all stages of cell cycle, being, nevertheless, more frequently use to detect metaphase chromosome imbalances and rearrangements or to operate with total DNA for probing in CGH analysis [2][3][4][5][6][7][10][11][12][13][14]. Further, we attempt to review each aforementioned approach in context of applications to single-cell chromosomal analysis.
FISH using wcp is a basis for MFISH (24-color FISH) and SKY [2,13,39,40]. These methods are valuable for cancer cytogenetics and, in some cases, for diagnosis of constitutional chromosome abnormalities [2,5,6,13,36]. For analysis of interphase chromosomes, MFISH/SKY is hardly applicable. Nevertheless, a study has visualized simultaneously all chromosomes in interphase nuclei of fibroblasts and prometaphase rosettes by 24-color MFISH [70]. Afterwards, such approaches have not been ever considered for related analysis. Two-to-five-color assays with wcp probes have been repeatedly used for molecular cytogenetic diagnosis of structural alterations to metaphase chromosomes [1-3,5-7,13,36,61] and  [14,54] investigation of genome organization in interphase nuclei [15,57,66,[70][71][72]. I-FISH with wcp probes is too problematic to be competitive with other techniques of interphase molecular cytogenetic diagnosis [7,10,33]. By microdissection of chromosomal loci for obtaining a set of probes that produce multicolor pseudo-G-banding, a high-resolution molecular cytogenetic technique for analysis of metaphase chromosomes termed MCB (multicolor banding) was proposed [43]. The latter has been consistently shown to be applicable for the identification of structural chromosome abnormalities and genome organization [2,13,36,43,45,61,73]. A modification of this technique, called recently interphase chromosome-specific MCB (ICS-MCB) that generates MCB of a homologous chromosome pair on single nuclei, has been demonstrated effective for studying human interphase chromosome organization and variations (somatic genomic variations and chromosome instability in health and disease) [3,7,10,13,19,23,24,[26][27][28][29]31,[33][34][35]74,75]. Apart from impossibility to analyze simultaneously several homologous chromosome pairs and relative complexity of the analysis, ICS-MCB does not possess major limitations. Moreover, this is the unique way to obtain a view on the entire interphase chromosome in its integrity [23,33,35].
The highest molecular cytogenetic resolution is achieved by fiber FISH (~2.3 kb) [52,76]. This approach was originally designed for mapping cloned DNA fragments at high resolution. The latter was found useful for investigation of genomic organization (on metaphase chromosomes), stalled transcription and genomic rearrangements (including large deletions within gene sequences) [51,52,76]. Although this technique is based on obtaining DNA fibers from interphase nuclei, it cannot be attributed to I-FISH. Singlecell molecular cytogenetic analysis by fiber FISH (especially, analysis of large cell populations) is highly complicated.

CGH
Since CGH compares quantitative differences between individual genomes, its applications are restricted to analysis of losses/gains of chromosomal (genomic) loci without direct visualization of chromosomes [4,77]. Array CGH can provide for a resolution up to nucleotide level, but still is poorly applicable for studying chromosomes of a cell. Nevertheless, several reports have demonstrated either standard CGH or array CGH on microdissected interphase nuclei to detect chromosome aberrations in single cells of preimplantation embryos [14,53,54]. Such approaches are applicable for unbalanced genomic rearrangements being useless for other areas of chromosome biology, which requires visualization of chromosomal DNA [10]. The potential of CGHbased single-cell analysis for molecular diagnosis and for surveys of somatic genomic variations remains to be estimated.

PNA and PRINS
Both PNA and PRINS can be successfully applied for studying human chromosomes [3,7,10,13,37,38]. PNAs are suggested to have several advantages over conventional molecular cytogenetic DNA probes, which are the result of their smaller size [38]. Notwithstanding, poor availability does not allow researchers to evaluate in situ hybridization with PNA probes for either metaphase or interphase molecular cytogenetics. Moreover, these probes are usually restricted to studying centromeric and telomeric repetitive chromosomal DNA.
In contrast to FISH and CGH, PRINS is based on another biochemical process (polymerase reaction) [37]. This makes it useful for case-control studies of newly discovered phenomena to exclude hypothetical errors that might be produced by hybridization [24]. Usually, PRINS shows almost the same results as FISH. Therefore, there is no apparent interest to substitute FISHbased techniques by PRINS, especially taking into account its essential limitation: available probes are oligonucleotides for pericentromeric/heterochromatic and few euchromatic regions (poorly reproducible!) [3,7,24,37].
The key process of all the studies aimed to analyze interphase chromosomes is visualization. In other words, lacking of direct (microscopic) DNA visualization makes all such researches incomplete. This becomes even more evident for studying chromosome organization in single cells. As one can see, only FISH-based techniques offer possibilities to detect either whole chromosomes or specific genomic loci of extremely small size in single cells. Therefore, to perform a valid study of human interphase chromosomes, I-FISH protocols are to use. The next part of our review addresses areas of I-FISH applications as well as its advantages and limitations.

I-FISH with wcp
It is generally recognized that FISH chromosomal painting using wcp is completely useless for identification of number and structure of interphase chromosomes (Figure 4) [3,7,10,13,33,35,80]. However, basic research of chromosome architecture in interphase is usually performed using I-FISH with wcp. These probes allows to visualize chromosome territories and their positioning relative to nuclear compartments ( Figure 4B) [57,[70][71][72]85,93]. For the last two decades, I-FISH-wcp approaches were almost the unique way to study genomic organization in interphase [72]. Some studies proposed to use the complete wcp set in an interphase MFISH reaction [70,93]. Nonetheless, these techniques are all limited in their abilities to paint chromosome territories (volumes) only (Table 2) [33].
There are several general problems that surround I-FISH application. As we have already mentioned, differences of hybridization efficiency complicate simultaneous applications of different probe sets [7]. For instance, signals of site-specific probes can be missed because of high brightness of wcp or centromeric probe signals. Here, the most apparent solution is ICS-MCB application [33,35]. However, some interphase protocols, mostly associated with molecular oncocytogenetics, are proven to be valid for diagnostic purposes [1,13,36,87,88]. DNA replication during S phase of cell cycle is another major problem of I-FISH applications [7,47]. Despite of recommendations concerning this type of I-FISH artifacts in the available literature, FISH analysis can be hindered by replicative signal appearance. This is mainly related to site-specific DNA probes, being, however, noticed during I-FISH with centromeric probes, as well [7,10,22,35,47] (Figure 6A-C). Additional source of numerous artifacts that can be considered as false-positive chromosome abnormalities in interphase is nuclear organization. In this context, the most problematic pattern of chromosome arrangement in the nucleus is related to chromosomal loci associations [94,95]. This significantly affects I-FISH results becoming even more important taking into account that numerous cell types are prone to exhibit intranuclear associations/pairing of genomic loci ( Figure 6D) [20,32,35,95]. Regardless frequent occurrence of related difficulties, the problem is easily solved by QFISH (Figure 6E) [23,24,28,32,35,95].
Finishing the list of interphase FISH-based techniques, it is to mention Immuno-FISH. This method combines immunohistochemical detection of proteins and FISH for visualization of DNA (RNA) targets [96][97][98]. Immuno-FISH is found applicable in cancer research/ diagnosis (simultaneous immunophenotyping and single-cell genetic analysis), studies of chromosome structure and organization, transplantation research, and identification of supramolecular disease mechanisms  [28,29,[96][97][98][99][100]. Figure 7 demonstrates Immuno-FISH used for studying interphase chromosomes in neuronal cells of the adult human brain [28,29]. After listing the most known methods of interphase molecular cytogenetics, it is to focus on their specific applications. Currently, there are there main biomedical areas requiring the use of I-FISH: analysis of intranuclear chromosome (genome) organization; identification of somatic (intercellular and intertissular) genomic variations; molecular cytogenetic diagnosis. Below, a brief description of these applications is given.

Genome organization in interphase
Spatial chromosome organization in interphase has been repeatedly shown to be a driving force for numerous crucial intracellular processes. It is suggested that specific arrangement of interphase chromosomes is likely to associate with genome activity, normal/abnormal cell  division, chromosome rearrangements occurring during meiosis and mitosis [7,[15][16][17]69,19,[70][71][72]75,93,100,101]. To get an integrated view of genome organization in interphase, numerous approaches should be applied. The leading role in these studies is played by I-FISH [7,80,72,93]. There could be several applications of I-FISH approaches for interphase chromosome analysis on this occasion: (i) identification of chromosome positioning and its relation to other nuclear compartments (nucleolus, Cajal bodies, nuclear speckles etc.) -I-FISH with wcp, interphase MFISH or ICS-MCB [19,31,23,33,35,34,[70][71][72]74,93]; (ii) studying correlation between positioning of specific genomic loci in relation to each other (i.e. association of whole chromosomes or their regions) and their behavior (transcriptional/replicative activity) for elucidating functional meaning of  nuclear organization and its driving forces -I-FISH with centromeric, site-specific and wcp, mFISH/QIFSH or ICS-MCB [7,19,31,23,33,35,32,34,57,66,69,72,74,75,[93][94][95]100]; (iii) analysis of chromosome behavior in relation to genome, epigenome and proteome changes for delineation of possible consequences of specific interphase chromosome architecture (i.e. occurring of somatic chromosomal mutations in cancers) -I-FISH with centromeric, site-specific and wcp, mFISH/QIFSH, ICS-MCB and Immuno-FISH [7,[15][16][17][18][19]34,69,71,72,74,75,[93][94][95]100,101]. Additional complication of I-FISH analysis of spatial chromosome organization is associated with structural preservation of nuclei. It is to note, that some researchers report about dependence of fixation type on I-FISH results [72,93], whereas others do not [71]. Regardless these debates, an alternative for I-FISH spatial genome analysis could be a suspension FISH (S-FISH) technique [102]. The advantage of this approach is related to possibility of studying threedimensional (3D) preserved nuclei from any human tissue, whereas other 3D preservation techniques require specific conditions of cell cultivation. The latter makes I-FISH to lose its main advantage. Together, it is to conclude that comprehensive description of functional significance of nuclear organization requires application of almost all known interphase molecular cytogenetic techniques.

Somatic genomic variations
During the last half decade, genomic variationsa source of human healthy and pathological diversityhave become a major focus of current biomedical research. Being involved in evolutionary and disease pathways, variations of the human genome are considered the main target of researches aimed to uncover disease mechanisms and species origins [103]. Soon after description of high rate of interindividual genomic diversification, it has been hypothesized that related processes-somatic genomic variationslie at the origin of intercellular genomic differences. Moreover, somatic variability of cellular genomes was proposed as a mechanism for complex human diseases [7,10,12]. The latter has been partially confirmed by high-resolution interphase molecular cytogenetic (molecular neurocytogenetic) studies of neurological and psychiatric diseases [7,[20][21][22][23][24][25][26][27][28][29]. The growing evidence for contribution of somatic genomic variations to the key physiological processes has been used for further hypothesizing about the emerging role of cell-to-cell genome variability in normal/abnormal human intrauterine development (including exogenous effects), cancerization, tissue-specific pathology (i.e. targeted neurodegeneration), sex differences in complex diseases, responses to molecular therapy of debilating neurological disorders [21,22,24,28,29,[104][105][106][107][108][109]. Altogether, this forms a basis for forthcoming researches in the field of single-cell biology. All these achievements were the result of numerous developments in interphase molecular cytogenetics. To prove it, we would like to refer to determination of stochastic (sporadic or background) aneuploidy level in human tissues (Table 3) [20][21][22][23][24]59,28,29,35,81,[109][110][111][112]. Looking through these data, it is hard to avoid the conclusion that aneuploidy rates become more reasonable if high-resolution I-FISH approaches are applied. Additionally, interindividual genomic variations can be detected in interphase by a parent-of-origin-determination FISH (pod-FISH) technique [113]. Together, I-FISH can be proposed as a required addition for studying genomic variations at microscopic and submicroscopic levels.

Molecular cytogenetic diagnosis
Molecular cytogenetic identification of chromosomal aberrations by I-FISH has been already mentioned in this review. Here, we would like to make some additional comments related to more specific problems of medical cytogenetics and to show again that studying chromosomes in interphase nuclei has profound effects on molecular cancer and prenatal diagnosis [114,115]. It is obvious that it is almost impossible to refer all the studies that used I-FISH. Here, we have preferred to describe several difficulties encountered during I-FISH introduction and usage for diagnostic purposes. Newly introduced interphase techniques (i.e. ICS-MCB) were used for research purposes only and, therefore, have not been tested for diagnostic validity. Despite of limiting practical application of these I-FISH protocols, related drawbacks can be easily eliminated by forthcoming studies. Another problem comes from the diagnosis of chromosomal mosaicism. There do not exist commonly accepted guidelines or criteria for mosaicism definition [7,10,35]. Regardless some attempts (for details see [35]), there is still no consensus concerning this topic. The Hopefully, similar studies have been already launched [20][21][22][23][24]59,28,29,35,81]. Finally, there are still no data or recommendations concerning correlation between metaphase and interphase diagnostic analysis of the same individual. In other words, it is still poorly understood what data is more valid. The structural point of view insists that metaphase analysis of chromosomes is more precise. From the other hand, mosaics require large cell populations to be analyzed. It becomes even more difficult to solve this problem when cases of complex, hidden (cryptic) or dynamic mosaicism are attempted to be described. Metaphase analysis in these case is indispensable for thorough definition of all cell lines, because simple I-FISH analyses are unable to precise a percentage of each cell line [116,117]. Moreover, some studies require additional data to obtain, i.e. parental origin of Figure 6 Problems of I-FISH with centromeric/site-specific DNA probes. (A) and (B) replication of specific genomic loci (LSI21 probe)some nuclei exhibit replicated signals, whereas in some nuclei it is not apparent; note the distance between signals can be more than a diameter of a signal; (C) asynchronous replication of a signal (DXZ1) in case of tetrasomy of chromosome X; note the difficulty to make a definitive conclusion about number of signals in the right nucleus; (D) Two-color FISH with centromeric/site-specific DNA probes for chromosome 1 shows chromosomal associations in a nucleus isolated from the adult human brain; note the impossibility to identify number of chromosomes; (E) QFISH demonstrating an association of centromeric regions of homologous chromosomes 9, but not a monosomy or chromosome loss (for more details see [32]).

Figure 7
Immuno-FISH. I-FISH using centromeric probe for chromosome Y (DYZ3) with immunostaining by NeuN (neuron-specific antibody) performed for the analysis of cells isolated from the human brain.
It is widely accepted that molecular cytogenetic diagnosis should be performed using a panel of techniques [1][2][3][4][5][6][7][8][9][10]. It could be either a combination of molecular cytogenetic techniques that use different platforms (i.e. FISH+CGH) or consecutive metaphase and interphase FISH analyses in cases of complex mosaics or balanced structural chromosome abnormalities. Thus, regardless significant developments in the field of molecular interphase cytogenetics, I-FISH techniques remain an addition to metaphase cytogenetics or whole genome screening approaches based on array CGH. The exception is few targeted assays for identification of known caner-associated translocations in interphase and preimplantation genetic diagnosis. Consequently, I-FISH should be more thoroughly analyzed in terms of the diagnostic potential to take a well-deserved place among genetic testing procedures.

Conclusions and future directions
Structural and behavioral properties of human interphase chromosomes in different tissue/cell types in health and disease remain largely unknown. To date, only fragmentary data on distantly related areas of interphase chromosome biology are available without an integral view of chromosome behavior and arrangement along cell cycle. An overview of molecular cytogenetic techniques for visualizing chromosomes in interphase evidences that a strong technological basis does exist for high-resolution analyses of chromosomes of almost all human tissues. Three main directions of I-FISH application has been advanced by developments in interphase molecular cytogenetics which has provided for possibilities to define functional consequences of spatiotemporal chromosome arrangement in the nuclei, to elucidate the role of such immense intercellular genomic diversity (somatic genomic variations), to propose new diagnostic solutions for medical genetics and oncology. I-FISH is the unique way to study variations and behavior of the genome in all the cell types of human organism, at all stages of cell cycle and at molecular and supramolecular resolutions. Thus, developments in interphase molecular cytogenetics open numerous prospects for genetics, cellular and molecular biology, genomic/molecular medicine. Taking into account data on technological aspects of studying human interphase chromosomes, we conclude that this biomedical direction has the potential to provide revolutionary solutions for basic and applied biomedical research in fields of human genetics and cell biology. This would be undoubtedly the result of combination of interphase molecular cytogenetic techniques (i. e. mFISH, QFISH, ICS-MCB, S-FISH, pod-FISH, Immuno-FISH etc), which has already given rise to several discoveries in current biomedicine.  [21,24,35] Fetal human brain mFISH/QFISH ICS-MCB~3 0% (~1.5% per chromosome) 35% + confined mosaicism [22,24,35] Blood I-FISH with centromeric probes Chromosome X: 1.5%-2.5% and 4.5%-5%*; Autosomes: 1.2% and 1% [110] mFISH/QFISH Chromosome X: 1.11%; Autosomes: 0,73% [25] Skin mFISH 2,2% and 4,4%* (whole genomeover 50%) [111] Liver mFISH~3% (whole genomeover 50%) [112] Adult human brain mFISH/QFISH ICS-MCB~1 0% (~0.5% per chromosome) [20,22,23,26,28,29]