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July/August 1999.Vol. 1.No. 5 invitededitorial

Molecular : Show me the colors

In this issue of in , 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 15 containing SNRPN will most likely have tification of marker , derivative chromosomes, an abnormal , 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 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 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 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 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 analyses have become much more routinely uti- abnormalities. These technologies run the gamut from using lized for the rapid prenatal detection of 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 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 probes, a 1 Mb BAC map for all chro- used to better understand , , 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 . 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?. 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 chromosomes. Combinatorial label- In sltu hybrldlwtlon wlth chromosome ,peiifir Ilhrarlri- Detection of rrlsomv ing was used and a unique pattern of karyotypic banding ?I and translocat~onof . Pro( i\'<~tlAa~tfSrr C'jA 1988;NJ:Y138-9142. 4 Cremer T, L~chterP, Borden I. \Yard DC, hlanuel~di\L. Deteit~onot chromosome involving different colors on each chromosome was gener- aberrations In and ~nterpha\etumor cells hv In 51tu hvhrldlzation usng ated. Other offshoots of this technology include a multi-color ihromosome apec~ficI~hrary probe,. Hurt] G'rllcr 1988:80:2ii-246. chromosome bar code and high resolution multi-color band- 5. Lichter P, Cremer T. Borden I, I\l.~nuel~d~sL. Ward DC. Dellnedtlon of lndivldual human chromosomes In metaphase and Interphase cell5 by In s~tusuppression ing, both of which allow the differentiation of the chromo- h\,bridlzation uslng chromosome speclfic I~hranprobes. HIIIIIGLIICI 1988:80.2?+234. some at specific region^.^',?^ As described above, all of these 6. Haddad BR,Schroik E, hleck I.Co\van I.lbungH. Ferguwn-Sm~thXIA. du Xlano~r methods can be utilized for a variety of studies. This includes 5, hed T. ldentlhiat~onof de noro chromosomal marker\ and der~\ati\esby Lpec- rral karyotpng. Hu~rrGrrlcr 1948;10:b194?. clinical cytogenetics (e.g., the determination of markers and 7. Huang B. Nlng Y,1.3mb AN. San~illnCI, lamehdor hl. Rled T, Bartley I. Idmtifica- de novo duplications), cytogenetics of neoplasia, radiation tlon of an unu\ual marker ihrornozome hv spectr.11 k.~rrotvp~ng.,4111 1 ~\InlGrrrct biology, cellular architecture, and comparative cytogenetics. 1998;80:368-372 8. Phelan hIC. Blaikhurn \\: Roger, KC'. Cra\\ ford EC, Cooley NK Ir. Schrock E. N~ng What is truly remarkable is not the advancement of mole- 1: R~edT FISH an.llva~a ot a cumple\ chromosome re.1rrangement ~n\ol\,ingnlne cular cytogenetic technology but its acceptance and absorbance bredpolnts on chronlosomea h. I?. I4 and I6 Prr.rror Ilrngrr 1998;lX:l 1 ?&I 180. into the clinical cytogenetics laboratories. These techniques 9. Sihrock E, \'eldman T. Pad~lla-NaahH, N~ngY, Spurheck 1, Ialal 5. Shaffer LG, Papenhausen P. Korrn~C. Phelan hlC. Kleldsen E. Schonherg SA. O'Brien P, Bieaecker have become much more routinely utilized to expand each L, du hlanolr 5. Kled T. Spectral karvotyplng refine, cvtopenetic d~agnost~csof ion- laboratory's capability to make proper diagnoses. The vast rt~tut~onalchromosomal abnorrnallt~es.HI~I~ G~-,lr.t 1997;101:?5-262, majority of laboratories in the United States currently have 111. Crolla 1A FISH and molecular studres of autosomal aupernumeral-r marker chro- mosomes eliluding those deri\red from . 11. Revle!\. of L~terature. some type of computerized FISH analysis system. More than ,4111 I hleii Grrrcr IYY8;75:367-381 70 laboratories already have a spectral karyotyping system. I I. Crolla TA. Long F. RnPeraH. Denn~sNR. FISH and molecular study of autosomal The big question for clinical cytogenetics does not involve how rupernunierdry marker chromosomes exclud~npthose derlred trom chrolnosome 15 and 22.1 Results of26 net\. caw A111 1Alr.d Gcr~c,tIYYX.;5:35566. the new technology should be used but what technology is 12. Schtvartr S. Deplnet T\V. Leana-Co\ I. IdaNB. Karaon Ehl. Park \'hI. Pasztor Lhl. necessary to use. It must also be decided when to use it, how Sheppard LC. Stallard R, \Z'olK Dl. Zlnn AB, Zuriher \'L. Zaiko~vsk~IL. Sex chro- to make it cost effective, and how to have the proper labor mosome markerb: Character~ratlonusing fluorescence in s~tuhyhrld17ation and revlrw of Ilter.lture. Ar~rI hlcri G~vlctIYY7;il:l-i effectiveness when performing the technique. As in all fields, 13. Vierhach K. Engels H. Gamerdlnger L'. Hansmann hl t>ellneat~onof sufernumer- technology does not come cheaply. It becomes incumbent ar). marker chromosomes In 38 patlent\. Am I ,\1c1f Go1t.r 1998;76:351-358. upon every laboratory to be able to integrate the technology 1.1. Lena-Cox I. lenk~nsL. Palmer CG. Plattner R. Shepp.lrd L. Flelter \\'L. zdikO,Vh!,l I. Thien F, Sch~vart75. hlolecular cytogcnet~canalys~s of in\ dup (15)chrolnosnlnei, and maximize its utilization, while still running a well-orga- using prohea apecific for the P~-~~der-\\'~ll~/An~elm.~n\vndro~ne regron. Cllnlial nized and fiscally responsive laboratory. implications. A111 I Hum Gt,?rcr 199-1:54:748-756

May/June/i999 ' Vol. 1. No. 4 July/August 1999 - Vol. 1 . No. 5

15. Lena-Cox I, Levln S, Sur.ln.l R, Wultaherg E, Keene (:I.. R.lftel LI, Sull~v.~nB. Schwdrtz An1 I Hlfnl Genet 1993;52:85&865. S. Chardctrr17~t1onof de novo dupll'.~t~~rn\In eight patlent\ hy ualng tlunrc\cence 22. Drward GW, Wyatt WA, Juneau AL. Carlson RO. Zinsmeister AR, lalal SM. Spurbeck In situ hyhrldlr~tlonw~th chro~iioromc-\pccltic LINA Ilhr~rle\.At11 I HIIIIIGCIICI IL, Sllvcr KT. Highly senslt~vefluorescence in situ hybridization method to detect 1993;5?:lO67-1073. double BCRIABL Fus~onand monitor response to therapy in chronic myeloid leukemia 16. Kuniar A. Hecker LA, [+net T\V. H.lrrn IM, Kurtz CL. Rohln NH, Cd~ldySLI. Wolff Mlc~od1998;91:3357-3365. Dl. Schw.lrtz S, hlolcculdr ch~r~1iter17at1un.lnd dcllne.~tlonof suhtlc deletlon5 In de 23. Spe~cherMR. Ballard SG, Ward DC. Karyotyplng human chromosomes by combl- novo "hC~ldnced"ihroniosnrn.~l re.lrr,ungement\. Hilt11 C;ctlc/ 19'18; 103.173- 178. natorial multi-fluor FISH. Nat Genet 1996;12:368-375. 17. S~rko-OaadraA, Caisldy SR, L)cp~netTM! Rohln NH. Llmwongsc C,Sihwdrtz S. MoI~ 24. Schrock E, du Mano~rS. Veldman T, Schoell B. Wienberg I, Ferguson-Smith MA, rcul.~rrefinement of k~ryotype:Bryund the c).togenrtli band. Gcrlcv~r\In Mni~ilnr. Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garin] Y, Ried T. Mult~colorspectral 1999:6: In prebs. karyotyplng of human chromosomes. Scrence 1996;273:494-497. 18. Ndtloni Cancer Inrtltutcr A rerourcc of arrdyed HAC Jones for ~dentihcdtlnnol 25. Muller S. O'Brlm PC. Ferguson-Smith MA, Wienberg J. A novel source of highly spe- cancer chromosome dbrrratlona. http:/lhacp~c.med.huH;~lo.eii~~/hum~~nioverv~e~v~htmlcific chromosome painting probes for human karyotype analys~sderlved from prl- 19. Kn~ghtSI. Horaley SW. Reyn R. L~wrlcNbI. Maher El, Cardy DL, Fllnt I. Kearney mate homologues. Hun1 Genet 1997;101:149-153. L. De\,elopnient and cllnicd appllcar~onof an Innovdtlve tluorescence In s~tuhybrldm- 26. Muller S. O'Brien PC, Ferguson-Sm~thMA, Wienherg I. Cross-species colour tlon tcchnlque whlch detccta suhmlcroscop~crearrangcmcnts lnvolvlng trlomeres. segmenting: A novel tool in human karyotype analysis. Cytometry 1998;33: Ellr I Hun1 Cerlct 1997;5:1-8. 445-452. 20. Fllnt I.\Vllkle AO. Buckle \'l,M'~nter RM, Holland AJ. h4cDrrm1d HE. The detection 27. Muller S, Rocchl M, Ferguson-Smith MA, Wienberg I. Toward a multicolor chro- of subtelomer~cchromoaonial rrarrangenientr In ldlopathlc mental retarddtion. Nat mosome bar code for the entlre human karyotype by fluorescence In s~tuhybrldiza- GI,I?~,~1995;9:132-140. tlon. H1rr71Genet 1997;100:271-278. ?I. \2'ard BE, Gersen SL, Care111 MP, MrGulre NM.Ddckowski WR, We~nste~nM. San- 28. Chudoha I, Plesch A, Lorch T, Lemke 1, Claussen U.Senger G. High resolut~onmul- dlin C. Warren R. Kllnger KM'. Rdp~dprenatal dlagnos~aof chromosomal aneuplol- t~color-bandlng:A new technique for refined FISH analysis of human chromosomes. dies bv tluorescence In sltu hybr~dlratlon:Cllnlcal experience wlth 4500 speclmena. Cytogmet Cell Genet 1999;84:156-160.

Coming in Genetics in Medicine September/October 1999 Issue

Original Articles Population-based studies reveal differences in the allelic frequencies of two functionally significant human interleukin-4 receptor polymorphisms in several ethnic groups Caggana M, Walker K, Reilly AA, Conroy JM, Duva S, Walsh AC Molecular refinement of karyotype: Beyond the cytogenetic band Sirko-Osadsa DA, Cassidy SB, Depinet TW, Robin NH, Limwongse C, Schwartz S Inconsistencies in genetic counseling and screening for consanguineous couples and their offspring: The need for practice guidelines Bennett RL, Hudgins L, Smith CO, Motulsky AG Exon lob of NF1 represents a mutatinal and harbors a recurrent missense mutation Y489C associated with aberrant splicing Messiaen LM, Callens IT; Roux KJ, Mortier GR, DePaepe A, Abramowicz M, Pericak-Vance MA, Vance JM, Wallace MR Transmission of by an affected mother Lossie AC, Driscoll DJ

Brief Revort Medium-chain acyl-CoA dehydrogenases deficiency: Sudden and unexpected death of a 45 year old Raymond K, Bale AE, Barnes CA, Rinaldo P Invited ReviewlCME Article Hearing loss Rehm H

180 Genetics IN ~ekine