Human molecular cytogenetics: From cells to nucleotides

Abstract

The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities 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 technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension 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 rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number 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 detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments 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: FISH; array-CGH; copy number variation; genomic disorders; molecular cytogenetics.

Figure 1

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

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).

Figure 3

Comparative Genomic Hybridization. (A) Conventional CGH analysis: a mixture of test DNA from a patient and a normal reference DNA labeled with different fluorochromes are hybridized to normal chromosome spreads (top panel). The left panel illustrates the hybridization pattern of chromosome 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.