An Overview of Cytogenetics

Bridget Herschap, M.D. 9/23/2013

Objectives }  History and Introduction of Cytogenetics }  Overview of Current Techniques }  Common cytogenetic tests and their clinical application }  Use of cytogenetic in the future

Introduction }  Cytogenetics is the study of structure, function, and

evolution of chromosomes. }  At the end of the 19th century the first tentative steps

were taken into the field of human cytogenetics. }  Cytogenetics has been used for many years for various

applications, from clinical diagnostics to basic genomic research.

Introduction }  In the ensuing years, human cytogenetics has been

transformed by technological advances that have combined innovations in molecular biology, chemistry and instrumentation.

}  The resolution and sensitivity of analyses have improved more than 10,000-fold in a very short time, first with the introduction of banding technology and later with fluorescence in situ hybridization (FISH).

}  Cytogeneticists can now extract far more information about the human genome than just chromosome number.

History }  Flemming published in 1882 the first drawn illustrations of

human chromosomes. }  In1888 the term “chromosome” (Greek for “stained

body”) was coined. }  Only a couple of years later, it was already anticipated

that chromosomes probably constitute the physical basis of heredity.

History }  Mammalian cell cultures were not yet possible and quality slide

were difficult to produce. }  Researchers had to deal with biopsies, tissue sections or

squashed tumor cell samples. }  Unable to obtain reproducible chromosome count }  Conflicting reports of chromosome number

}  Von Winiwarter counted a different number in males (n = 47) as in females (n = 48).

}  Painter published in 1923 several studies of a chromosome number of 48 based on a study of meiotic chromosomes in several testis biopsies taken from incarcerated, castrated males who had been sentenced to death.

Breakthrough }  Cells kept for several minutes in a hypotonic salt

solution instead of an isotonic one before fixation gave a much better spreading of the chromosomes. }  Ordinary water accidentally applied instead of a salt solution

for washing the cells.

}  The addition of colchicine to cell cultures captured the cells in their metaphase stage.

}  Subsequently, the cells are not able to finish mitosis and an increasing number of metaphases become available for microscopic studies.

Tjio and Levan } “Before a renewed, careful control has been made of the chromosome number in spermatogonial mitoses of man we do not wish to generalize our present findings into a statement that the chromosome number of man is 2n = 46, but it is hard to avoid the conclusion that this would be the most natural explanation of our observations."

History }  Human cytogeneticists were dealt a good hand by

evolution. }  Fortunately, differences in the relative size of human

chromosomes and the position of the centromeric constriction allowed cytogeneticists to match up the 23 pairs and classify them into seven groups (A to G) with relative ease

History: BCR-ABL }  1960: The first acquired (not constitutional) chromosome

abnormality, the “Philadelphia chromosome”, was recognized in bone marrow cells of a patient suffering from chronic myeloid leukemia.

}  13 years before it became apparent that this was not a simple deletion but the result of a reciprocal translocation between the chromosomes 9 and 22.

}  After almost another 13 years, it was discovered that this translocation resulted in a new fusion gene between BCR and ABL.

Cytogenetics: from the bench to the clinic

}  1966: Cells cultured from amniotic fluid could be used to determine the chromosome content of the fetus. }  This is the technique that is still most widely used for prenatal

chromosome studies.

}  Studies of abortions and miscarriages revealed numerical aberrations in over 50% of abortions. }  Resulting in a tetra- or triploidy, a trisomy (e.g., chromosomes

13, 18, 21, and often 16) or monosomy (45,X).

History }  Until the early 1970s, all cytogenetic studies were

performed on “solid” stained chromosomes which hampered identification of individual chromosomes and the detection of most of the structural aberrations.

}  Therefore, many efforts were made to develop a technique for a clear discrimination between the various chromosome pairs.

}  For routine use in a clinical setting, the G-banding technique based on the application of trypsin followed by Giemsa staining became the most accepted method worldwide.

History }  Resolution of chromosome studies remained relatively

limited with an approximate count of 500 bands per haploid genome.

}  However, synchronizing lymphocyte cultures, lead to increased number of cells being in pro-metaphase or even prophase stage instead of metaphase. }  In these much longer chromosomes, regular metaphase bands

appeared to split-up in many sub-bands, thus increasing resolution from 500 to over 1000 bands in a haploid genome (6 million base pairs ≈ 50 genes per band).

High Resolution Banding

Fig. 4. High resolution banding of chromosomes 15 from 3 different patients with Prader Willi syndrome. From left to right, the ideogram of a normal #15, than in the middle the normal #15 while on the right side the #15 with a deletion of the most proximal band is shown. Both the normal and abnormal #15 are from the same cell.

Fig. 3. Balanced translocation between chromosomes 11 and 22. At the right side of the picture are the chromosomes 11 and 22 of a normal metaphase cell, while the left side shows high resolution chromosomes from a prometaphase. The aberrant chromosomes are presented as der(11) and der(22).

An Overview of Cytogenetics

Microscope to Molecule

Microscope to Molecule }  Once a rearranged chromosome has been identified, the

next step is to position the translocation breakpoints or deletion boundaries relative to genes on molecular maps.

}  This step can be accomplished by using techniques that physically separate abnormal and normal chromosomes so that they can be independently assayed for gene content.

}  Three methods have been particularly useful in achieving this: }  somatic-cell-hybrid technology }  fluorescence-activated cell (chromosome) sorting (FACS) }  FISH

FISH

}  Fig. 6. Various applications of FISH. (a) Simultaneous chromosome paints of the X-chromosomes (pink) and their centromeres (yellow). (b) Interphase FISH with probes specific for the Y-chromosome (red) and the X-chromosome centromere (blue) on uncultured amniocytes of a fetus with a 47,XXY karyotype. (c) Detection of a submicroscopic deletion in the long arm of a chromosome 22 in a patient with DiGeorge syndrome. The normal chromosome 22 shows two signals (one from the DiGeorge region and one from the tip of the long arm as a control signal), while at the second chromosome 22, only the control signal appears to be present since the DiGeorge region is absent (arrow). (d) Spectral karyotyping (SKY) of the complete chromosome complement, where all chromosome pairs have their own (artificial) color.

FISH }  Despite the development of high-resolution chromosome

banding, numerous patients still showing clear clinical symptoms were found to have no visible aberrations at the cytogenetic level.

}  The next advance to revolutionize cytogenetics, FISH, provided a direct link between microscope and sequence.

FISH }  Each probe is a cloned piece

of the genome that is conjugated to a reporter molecule, such as biotin.

}  After denaturation, the probe is allowed to seek out its complement in the chromosomal DNA, and these locations are then marked with a fluorescent reagent, such as avidin-FITC, that binds to the reporter attached to the DNA probe.

Molecular cytogenetics }  The gap between cytogenetics and molecular studies became

smaller. }  As molecular biologists filled in the genome maps, large

collections of molecular reagents in the form of cloned, mapped segments of the human genome (cosmids, BACs, PACs, and YACs) became available with which abnormal chromosomes could be characterized by FISH to identify affected genes.

}  For example, FISH analyses identified clones that cross the two breakpoints of the pericentric inversion of chromosome 16 seen in patients with AML.

}  This finding set the stage for the identification of the two genes (MYH11, smooth muscle myosin heavy chain 11, and CBFB, the -subunit of core-binding factor) that, when aberrantly fused, cause the leukemic transformation.

}  Even more importantly, FISH opened up the nuclei of non-dividing cells to karyotype analysis.

}  Conventional cytogenetics requires the capture of cells in mitosis, and many samples, particularly those from solid tumors, produce few, if any, analyzable metaphases.

Interphase FISH }  Because DNA is packaged

10,000-fold more loosely in interphase nuclei than in metaphase chromosomes, abnormalities that are not resolvable by metaphase FISH can be detected by interphase FISH.

}  Shifts in relative spot position reveal structural rearrangements, such as translocations and inversions.

Interphase FISH }  Interphase analysis is also useful when cells are rare, such

as in minimal residual disease or micrometastasis. }  Interphase FISH with centromere probes also allows the

rapid screening of large numbers of cells to identify chromosomal instability in tumour cell nuclei.

}  a | Outline of the spectral karyotyping (SKY) protocol. SKY and multicolour fluorescence in situ hybridization (M-FISH) differ only in the method used to measure the spectral characteristics of each pixel in the image (see main text). Cot-1 DNA is enriched in repetitive sequences, and by binding to repetitive sequences in the fluorescently tagged probes, it suppresses their hybridization to target chromosomes. b | The application of SKY to normal interphase and metaphase human cells; the highly rearranged karyotype of a bladder cancer cell is shown in c. Arrows point to inter-chromosomal rearrangements.

CGH: a surrogate for chromosomes }  The next transformation of cytogenetics came with the

realization that genome-wide scans for the loss or gain of chromosomal material could be conducted without even looking directly at the subject's chromosomes. The technique that made this possible is called comparative genome hybridization (CGH).

CGH: a surrogate for chromosomes }  Metaphase chromosomes are replaced as the target by large

numbers of mapped clones that are spotted onto a standard glass slide. }  increased the resolution of screening for genomic copy number gains

and losses

}  In array CGH, the test and normal reference genomes, which are used as probes, are differentially labelled and co-hybridized to a microarray.

}  The array is then imaged and the relative fluorescence intensities are calculated for each mapped clone, with the resulting intensity ratio reflecting the DNA copy number difference.

}  The resolution of the analysis is restricted only by clone size and by the density of clones on the array.

CGH }  Important in cancer cytogenetics, in which it is used to

identify chromosomal regions that are recurrently lost or gained in tumors. }  For example, CGH led the way to the identification of PIK3CA,

the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), as an oncogene in ovarian cancer.

}  DNA-amplification techniques have also been developed to find genetic alterations in small samples of rare cells, such as rogue cells found in blood that have escaped a primary tumor and might foreshadow metastasis.

Limitations }  For rearrangements that do not involve genomic

imbalances, such as balanced chromosome translocations and inversions, the use of CGH is limited.

}  In addition, whole-genome copy number changes (ploidy changes) cannot be detected.

}  Furthermore, CGH provides no information about the structural arrangements of chromosome segments that are involved in gains and losses.

}  Despite these limitations, CGH has become one of the most widely used cytogenetic techniques in both basic research and molecular diagnostics.

Future of CGH }  These analyses should generate prognostic markers,

identify new tumor-suppressor genes or oncogenes and, ultimately, lead to a better understanding of the cancer process.

}  It is hoped that technological advances, such as array-CGH, will reduce the time and cost of cytogenetic analyses so that they can be accessed by more families.

Conclusion }  Although cytogenetics is moving into the direction of

molecular approaches instead of microscopy, these developments by no means imply that the “old fashioned” banding techniques practiced for over 30 years now will disappear from the laboratories.

}  Karyotyping remains the single (and simple) technique to obtain a quick overview of the complete human genome.

Conclusion }  Cytogenetics continues to reinvent itself to aid explorations of

chromosome structure, function and evolution. }  Banding techniques, which are unchanged from the 1970s apart

from the introduction of digital image handling, are now combined with state-of-the-art multicolour FISH and molecular analysis.

References

}  Albertson, D. G. et al. Chromosome aberrations in solid tumors. Nature Genet. 34, 369–376 (2003).

}  BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409, 953–958 (2001).

}  Dominique F.C.M Smeets, Historical prospective of human cytogenetics: from microscope to microarray, Clinical Biochemistry, Volume 37, Issue 6, June 2004, Pages 439-446, ISSN 0009-9120, http://dx.doi.org/10.1016/j.clinbiochem.2004.03.006. (http://www.sciencedirect.com/science/article/pii/S0009912004000864)

}  Takagi N. and Sasaki M, A phylogenetic study of bird karyotypes. Chromosoma 21. V. 1974, Volume 46,pp 91-120

}  Speicher MR, Carter NP. The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet. 2005 Oct;6(10):782-92.

}  Zitzelsberger, H. F., O'Brien, B. & Weier, H. U. G. in FISH Technology (eds Rautenstrauss, B. & Liehr, T.) 408–424 (Springer, Heidelberg, 2002).