Human interphase chromosomes: a review of available molecular cytogenetic technologies

Abstract

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.

Figure 1

Two- and three-color I-FISH with centromeric DNA probes. (A) normal diploid nucleus with two signals for chromosome 1 and chromosome 15; (B) monosomic nucleus with two signals for chromosome 1 and one signal for chromosome 15; (C) trisomic nucleus with two signals for chromosome 1 and three signals for chromosome 15; (D) normal diploid nucleus with two signals for chromosome 1, chromosome 9 and chromosome 16; (E) monosomic nucleus with two signals for chromosome 1 and chrosmome 9 and one signal for chromosome 16; (F) trisomic nucleus with two signals for chromosome 1 and chromosome 16 and three signals for chromosome 9; (G) triploid nucleus with three signals for chromosome 16 and chromosome 18; (H) tetraploid nucleus with two signals for chromosome X and chromosome Y; (I) tetraploid nucleus with two signals for chromosome X and chromosome Y, and four signals for chromosome 1.

Figure 2

I-FISH with site-specific DNA probes. (A) normal diploid nucleus with two signals for chromosome 21; (B) trisomic nucleus with three signals for chromosome 21; (C) interphase nucleus exhibiting co-localization of ABL and BCR genes probably due to t(9;22)/Philadelphia chromosome.

Figure 3

Five-color I-FISH (mFISH) with DNA probes for chromosomes 18, X and Y (centromeric probes) as well as 13 and 21 (site-specific probes). a presumably normal (diploid) male nucleus isolated from the adult human brain.

Figure 4

I-FISH with two wcp for chromosomes 7 and 21. (A) ambiguous chromosome territories provide information neither about number of chromosomes nor about structure of chromosomes (chromosome 7 -- green signal; chromosome 21 -- red signals), whereas this individual presented with regular unbalanced t(7;21); more details are given in Vorsanova et al. 2008 [64]; (B) chromosome territories in an interphase nucleus of a cell isolated from the ataxia-telangiectasia brain (chromosome 7 -- green signals; chromosome 14 -- red signal); note the impossibility to identify number of chromosomes 14.

Figure 5

ICS-MCB with chromosome 21-specific probe. Monosomy (loss) of chromosome 21 in a nucleus isolated from the Alzheimer's disease brain.

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.