July/August 1999.Vol. 1.No. 5 invitededitorial Molecular cytogenetics: Show me the colors In this issue of Genetics in Medicine, an article by Jalal and the field of cytogenetics a much more vibrant and fruitful Law entitled: "The Utility of M-FISH in Clinical Cytogenet- endeavor, allowing us to unequivocally identify marker chro- ics" is presented.' The authors examine the utilization of multi- mosomes that are found both prenatally and postnatally. It color FISH (M-FISH) in clinical cytogenetics by studying seven has allowed distinct phenotypic correlations to be made for cases, six of which were cytogenetically abnormal, to deter- many specific markers, specifically those derived from chro- mine the efficacy and utility of this technology. They have mosomes 12, 15, 18, and 22. For example, a marker derived shown that this multi-color technology is useful for the iden- from chromosome 15 containing SNRPN will most likely have tification of marker chromosomes, derivative chromosomes, an abnormal phenotype, whereas the marker without SNRPN and in the analysis of complex karyotypes. In addition, they will more likely have a normal phen~type.'~-'~FISH can be illustrate limitations of this technology and its inability to detect used to determine the origin of extra unidentified material on some specific abnormalities. They also compare its limitation derivative chromosomes and single copy probes can deter- to the similar technology of spectral karyotyping (SKY).This mine the extent of rearrangement.15 Both subtle and complex article follows a similar article in the inaugural issue of Genet- rearrangements can be elucidated by a variety of methods. ics in Medicine (Volume 1) in which Levy and her colleagues Using a series of YACs, we have delineated subtle deletions in described the utilization of comparative genomic hybridiza- several cytogenetically "balanced" translocations and have elu- tion (CGH) to study 12 abnormal derivative chromosomes cidated known genes that are either deleted or present in indi- (five markers, five unbalanced translocations, and two intra- viduals with cytogenetic deletions.16-I' One of the more chromosomal duplications), highlighting the utility of this common uses of FISH, and by many accounts one of the technology also to identify unknown chromosomal material.' important aspects, is in the identification of microdeletion The study of chromosomes has a relatively short history, syndromes. These studies have taught us that the frequency and since Tjio and Levan first identified the correct chromo- of many abnormalities may be greater than we initially imag- some number of 46 in humans in 1956, there has been con- ined. For example, the frequency of deletions of chromosome stant improvement and refinement of the technologies that are 22 may be as high as 1:3000. The National Institutes of Health routinely used for chromosome identification. Analysis of chro- initiative to create a FISH-BAC map, with markers one mosomes using solid staining progressed to identification by megabase apart on every chromosome, provides the oppor- banding in 1971, which was followed shortly thereafter ( 1976) tunity to precisely define structural rearrangements.18 We will by utilization of high-resolution technology. At that point, be able to determine the precise amount of material on acces- advances in cytogenetics came to a standstill and questions sory marker chromosomes and to identify small deletions in concerning the overall efficacy of cytogenetics and its useful- apparently balanced translocations. We have already shown ness in the future began to be posed. However, the future applic- this phenomenon utilizing YACs, but it will be more effica- ability of cytogenetics became clearly delineated and apparent cious with BACs. with the ground breaking experiments of Pinkel and Gray and With the identification of unique subtelomeric regions on of Ward and his colleagues who laid the groundwork in 1988 each individual chromosome arm, studies can be done answer- for molecular cytogenetics, with technology revolving around ing whether, and if so to what extent, subtelomeric variation the utilization of fluorescence in situ hybridization.'-5 is clinically important. Both cryptic rearrangements as well Today, cytogeneticists have an arsenal of technologies that as cryptic subtelomeric deletions have been associated with can be used both clinically and from a research perspective to idiopathic mental retardati~n.",'~As the technology increases, better understand chromosome structure and function. These it is extremely likely that multicolor telomeric probes will be techniques can be used to make chromosome identification, available in which all of the chromosome arms can be rou- to study the mechanism of chromosomal aberrations, and to tinely analyzed in appropriate cases. better understand the phenotypic effects of chromosomal Interphase analyses have become much more routinely uti- abnormalities. These technologies run the gamut from using lized for the rapid prenatal detection of aneuploidy or for the single chromosome painting probes to identify one specific detection of a Bcr-Abl fusion in chronic myelogenous leukemia. chromosome to using single copy probes to look for specific A large number of prenatal laboratories are currently doing deletions or duplications of material. Comparative genomic prenatal interphase analysis, to a limited degree, to rule out hybridization can be utilized to better analyze neoplasia and, aneuploidy of chromosomes 13, 18,21, X, and Y.l' Its appli- as discussed in the article by Jalal and Law presented in this cations in cancer cytogenetics have vastly multiplied, in which issue of the journal, multicolor FISH or SKY can be used to probes for several different site-specific translocations have analyze marker chromosomes, derivative chromosomes, and been developed. These probes, such as those developed for complex karyotype~.~,"~All of these technologies have made detecting the Bcr-abl rearrangement in CML, can not only be 178 Genetics IN ~e&be July/August 1999 - Vol. 1- No. 5 effectively used for studying interphase cells, but have become In all respects, the future continues to remain bright for extremely important for monitoring the effectiveness of treat- molecular cytogenetics. Techniques continue to be tested and ments.??The utilization of molecular cytogenetics has vastly to ultimately find their proper place in the clinical laboratory. expanded not only in the clinical realm but also in the research As work continues, we will see the future development of area. Here, this technology has become much more routinely multi-color telomere probes, a 1 Mb BAC map for all chro- used to better understand meiosis, cell, and nucleus architec- mosomes, and comparative genomic hybridization with an ture, and in the identification of mouse chromosomes, espe- array technology that might allow for the rapid detection of cially in the creation of embryonic stem cells. both deletions and duplications within the genome. These The newest FISH technologies are the multi-color kary- developments should ultimately provide the opportunity to otyping techniques as described by Jalal and Law in this issue.' clearly delineate all abnormalities on the molecular level. This Three major different types of multi-color FISH are available: will provide detailed phenotype-karyotype for detecting M-FISH, SKY, and Fk-FISH. M-FISH was first described by abnormalities, allowing both prenatal and postnatal progno- Spiecher et al. in 1996.'' This technique is based on a combi- sis of these chromosomal aberrations. All of these technolo- natorial labeling approach in which six different tluorochromes gies are continually being tested and absorbed within the are utilized in combination, ylelding a possible 63 combina- clinical laboratories, which ultimately must determine the best tions (2"- 1). Using these fluorochromes with optical filters way to diagnose patients and to determine how this technol- between 350 and 770 nm, they visualized 27 combinatorially ogy can best be successful. labeled probes simultaneously. These were analyzed using sophisticated software allowing each individual chromosome Stuart Schwartz, PhD Department of Genetics and to be pseudocolored. In the same year, Schrock et al. ?" reported Centerfor Htl~nnr~Getzetics multi-color karyotyping that was interferometer-based spec- University Hospitals of Cleveh~nd tral imaging, in contrast to the fluorochrome based system Clevelnrzd, Ohio described above. They used an interferometer to generate a fluorochrome-specific optical path difference that provides References spectral information. In conjunction with a CCD camera, the I lalal Shl, Law, hlE. Utllln. ot hl-F~ahIn clinical cvtogenetlih. Gcrletric 111 .\ILY~ICIIIP tluorescence emission spectrum can be recovered simultane- 1999:1:181-186. ously at all points. Muller et al., in 1997, proposed using cross- 2. Levy B. Dunn Thl, Kaffe S. Kardon N. Hlrschorn h: Clinical appl~iat~on\of com- paratlve genomlc hybr~d~zat~on.L'rrlrtro 111 Afedri-rrlc 1YYX.l +I?. species multi-color banding (RX-FISH),?~.'~utilizing probes 3. Pinkel D, Landsgent I. Collins C, Fusioe I. Segraves K. Luca, I. Grav PV. Fluorescenie from flow-sorted gibbon chromosomes. Combinatorial label- In sltu hybrldlwtlon wlth human chromosome ,peiifir Ilhrarlri- Detection of rrlsomv ing was used and a unique pattern of karyotypic banding ?I and translocat~onof chromosome 4. Pro( i\'<~tlAa~tfSrr C'jA 1988;NJ:Y138-9142. 4 Cremer T, L~chterP, Borden