Cytogenet Cell G enet 6 0 :257-269 (1992)

Abstracts of presentations at the

Thirtieth Annual American Cytogenetics Conference held on March 15-18, 1992

at the Virginia Beach Resort Hotel and Conference Center Virginia Beach, VA



Received for publication March 20, 1992

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Arthur Brothman (C onference Chair) Peter Jacky (Program Chair) Linda Cannizzaro Janet Cowan Susanne Gollin Stan Hoegerman Patricia Howard-Peebles James Mascarello Nagesh Rao Debra Saxe S tuart Schwartz Patrick S torto U rvashi S urti John W iley

C ontent

(Abstracts are published within the sections of the program in the order of their presentation, with the exception of Invited Presentations which are listed first.)


"DNA SEQUENCE SIGNATURES AND CHROMOSOME ORGANIZATION".............................................................................. 1

Beatrice Mintz


David Page


"SEX DETERMINATION AND TURNER SYNDROME: WHAT HAVE WE LEARNED SINCE 1 9 5 9 ? ".......................................... 3 1:





Moderators: AR Brothman and MJ Pettenati A TECHNICAL OVERVIEW OF FLUORESCENCE IN SITU HYBRIDIZATION. (No abstract) LS J e n k in s .................................................................................................................................................... ANALYSIS OF ACCESSORY MARKER CHROMOSOMES USING STANDARD AND MOLECULAR CYTOGENETIC TECHNIQUES. L S Jenkins, L Bros, S Rosenfeld, R Bachman, M Lipson, D Witt, and J M a n n ....................................................................................................................................4 LOCALIZATION OF COSMIDS TO CHROMOSOME 22 BY FLUORESCENCE IN SITU HYBRIDIZATION. B Sellinger, YO Tatsumura, M Rasmussen, H Vissing, B S Emanuel, and ML Budarf................................................................................................................................................. 5 THE USE OF FLUORESCENCE IN-SITU HYBRIDIZATION IN THE CHARACTERIZATION OF CYTOGENETIC ABNORMALITIES. AR Brothman, AM Patel, PD Storto, PF Chance................ 6 USE OF CHROMOSOME SPECIFIC FLUORESCENT PROBES IN CUNICAL CYTOGENETIC STUDIES. G Hicks, M Law, E Christensen, J Spurbeck, and SM J a la l................................... 7 CYTOLOGICAL IDENTIFICATION OF TELOMERIC SEQUENCES AT TERMINAL REGIONS OF CHROMOSOME DELETIONS THROUGH FLUORESCENT IN SITU HYBRIDIZATION (FISH). N Rao, R Hayworth, MJ Pettenati.......................................................................................................... 8

AN APPARENTLY BENIGN EUCHROMATIC DUPLICATION IN CHROMOSOME 9: CYTOGENETIC AND MOLECULAR CHARACTERIZATION. L Charity, G Stetten, C Rosenberg and J S tarnberg...............................................................................................................................................1 0 SUPERNUMERARY i(5P) IN A CHILD AND A DIFFERENT MARKER CHROMOSOME PRESENT IN HER MOTHER. W Stanley, K Rosenbaum, G Devine, T Ellingham and B M urphy........ 1 1

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A CASE OF DEL 1q42.3 DUE TO A DE NOVO NONRECIPROCAL TRANSLOCATION (1 ;8) CONFIRMED BY FLUORESCENT IN SITU HYBRIDIZATION. JC Stout, J E Wiley, T Kushnick.................................................................................................................................................... 9

Abstracts - 30th Annual American Cytogenetics Conference






M oderators: MM Cohen and PB Jacky CYTOGENETIC AND MOLECULAR ANALYSIS OF NON-DISJUNCTION IN TRISOMY 21. BJ Lorber, M Grantham, J Peters, HF Willard, and T H a sso ld ................................................... 1 2 X-INACTIVATION PATTERNS IN MULTIPLE TISSUES OF THREE CASES OF APPARENTLY BALANCED X/AUTOSOME TRANSLOCATIONS WITH ABNORMAL PHENOTYPES. JL Zackowski, ES Cantu, DJ Driscoll, H Ostrer, RT Zori, BA G ra y ........................................... 1 3 PARACENTRIC INVERSIONS IN MAN: A REVIEW OF 431 CASES. MJ Pettanati and N Rao......................................................................................................................................................... 14 CHROMOSOME BREAKAGE IN HUMAN SPERM. AM Estop, F Levinson, and S Munne........ 1 5 METAPHASE PREPARATION FROM CULTURES ARRESTED IN G2 BY TOPOISOMERASE INHIBITION. JF S to n e ............................................................................................................................ 16 PRENATAL CYTOGENETIC DIAGNOSIS OF THE FRAGILE X CHROMOSOME: FEASIBILITY AND SPEED OF IN SITU CLONAL METHOD IN AMNIOTIC FLUID CELL TISSUE CULTURE. LR Shapiro, PL Wilmot, and LE Andree..............................................................................................1 7 THE NUMBER OF CELLS ANALYZED TO DETECT MOSAICISM. J Zenger-Hain, D Kramer, F Myrick, and D L Van D yke................................................................................................................1 8 THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION TO DETECT SPECIFIC CHROMOSOMES IN UNCULTURED FETAL CELLS. G Prabhakar, K J Blakemore, V L Jaswaney and G Stetten........................................................................................................................ 19 HYBRIDIZATION OF TELOMERIC DNA TO INTERSTITIAL CHROMOSOMAL SITES: CORRELATION WITH FRAGILE SITES AND BREAKPOINTS OF CHROMOSOMAL REARRANGEMENTS. S Adekunle, HE Wyandt and A M ilunsky.................................................2 0 FRAGILE X CYTOGENETIC GUIDELINES REVIEWED: RELEVANCE TO FRA(X) MOLECULAR GENETIC STUDIES AND TO THE APPROPRIATENESS OF CHROMOSOME STUDIES IN ALL CASES OF DEVELOPMENTAL DELAY. A DISCUSSION. PB Jacky and EC Jenkins................ 2 1





Moderators: WR Breg and JT Mascarello ANEUPLOIDY SECONDARY TO GENE TARGETING IN EMBRYONIC STEM CELLS. JT Mascarello, B Koller and O S m ith ie s............................................................................................2 2 CHROMOSOMAL RELATIONSHIPS BETWEEN PEROMYSCUS OREAS AND PEROMYSCUS MANICULATUS ARTEM ISIAE. LR Smith and IF Greenbaum.................................................... 2 3 CHROMOSOMAL FRAGILE SITES IN THE DEER MOUSE: DISTRIBUTION, VARIATION, AND POTENTIAL ROLE IN CHROMOSOMAL EVOLUTION. BF McAllister and IF Greenbaum....... 2 4

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Abstracts - 30th Annual American Cytogenetics Conference






CYTOGENETICS AND ORIGINS OF OVARIAN GERM CELL TUMORS. L Hoffner, R Deka, RE Ferrell, A Chakravarti and U S u rti.............................................................................................. 2 6 CONSISTENT NUMERICAL CHROMOSOME ABERRATIONS IN CONGENITAL FIBROSARCOMA. SM Gollin, S Sankary, E Wiener, W Robichaux, WP Swaney, PS Malone, and PS Dickm an.............................................................................................................................................. 2 7 THE RECURRENT CHROMOSOME CHANGES IN SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK REGION. DL Van Dyke, MJ Forsham, MJ Benninger, TE C a re y..........................2 8 DO CHROMOSOME ABNORMALITIES PREDICT MALIGNANT BEHAVIOR IN MENINGIOMAS? BF Schneider, SR Vandenberg, KW Sudduth and WL G olden...................................................2 9 DEFINING THE BREAKPOINT IN RENAL CELL CARCINOMA USING CHROMOSOME MICRODISSECTION. L Boghosian-Sell, LA Cannizzaro............................................................-.3 0







Moderators: S Schwartz and PN Howard-Peebles UTIUZATION OF FLUORESCENCE IN SITU HYBRIDIZATION FOR DELINEATION OF PERICENTRIC CHROMOSOME BREAKPOINTS. S Schwartz, J Leana-Cox, C Punzalan and B S u lliv a n ................................................................................................................................................3 1 UTIUZATION OF CHROMOSOME-SPECIFIC DNA UBRARIES TO DELINEATE THE ORIGIN OF DE NOVO DUPLICATIONS. J Leana-Cox, S Levin, R Surana, J Zackowski, and S Schw artz............................................................................................................................................... 3 2 PARTIAL DUPUCATION 9q AND DELETION 6q IDENTIFIED BY CHROMOSOME PAINTING. N Spinner, A Schneider, E Zackai, W Golden, and J Lucas.......................................................... 3 3





Moderators: L Cannizzaro and U Surti ANALYSIS OF LEUKEMIA-ASSOCIATED CHROMOSOMAL REARRANGEMENTS WITH CHROMOSOME-SPECIFIC DNA LIBRARIES AND FLUORESCENCE IN SITU HYBRIDIZATION. BA Sullivan, J Leana-Cox, C Schiffer, and S S chw artz........................................................... 3 4 DUPLICATION OF 3q21->q26 IN A PATIENT WITH M1 ACUTE MYELOGENOUS LEUKEMIA (AML). A Scalise, C Theodossiou, N Wisch, V N a jfe ld ..............................................................3 5

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LOSS OF THE Y-CHROMOSOME FROM BONE MARROW CELLS: IS IT AN AGING PHENOMENON OR A MALIGNANCY MARKER? S Shekter-Levin, ME Sherer, N Wald, and SM G ollin .............................................................................................................................................. 3 6


Abstracts - 30th Annual American Cytogenetics Conference

D NA SEQUENCE SIGNATURES A N D CHROMOSOME ORGANIZATION Laura Manuclidis, Yale Medical School. N ew H aven, Connecticut

The regu lation o f g e n e tic a c tiv ity is fu n d am en tal for the d evelop m en t of com plex organism s. S elected sets of gen es on different chrom osom es must be sequ en tially and appropriately recruited. It has becom e apparent that different repeated D N A sequence m otifs are nonrandomly clustered in the genom e, and are associated with different classes of genes. Furthermore, these repeats span DNA lengths of 0.1 to -9M b, and are often associated with different tim es of replication. It is likely that su ch larger features o f seq u en ce org a n iza tio n h ave a role in the transcriptional activation (or silen cin g ) o f d ifferen t sets o f gen es in interphase. Different com b ination s o f repeats m ay sp ecify individual d om ain s of a related set. Several m ech an ism s m ay be u tilized in this setting. 1) Repeated sequence m otifs m ay provide recognition signals for the com plex m olecular machinery in volved in g en e regulation. 1 will discuss endogenous retroviral elem ents in this context. 2) Distinct types of structural conformations may also be designated by repeated D N A s. Notably, insertion of a very large transgenic tandem repeat resu lts in a heterochrom atic configuration for this locus in interphase. 3) Selected repeats may also have a role in determ ining chrom osom al position s in different cell types. Threedim ensional studies of interphase nuclei to date are consistent with this concept. With recent refinem ents for in-situ hybridization and computer analysis it is now possible to experim entally address more subtle structural parameters and potential functions of non-coding sequences.

3 SEX DETERMINATION AND TURNER SYNDROME: WHAT HAVE WE LEARNED SINCE 1959? D. C. Page, R. Alagappan. P. Beer-Romero. M. Behlke, J. Bogan, L. Brown, A. Cheng, S. Foote, T. Lee, S.-W. Luoh, J. Pringle, E. Simpson, D. Vollrath, and A. Zinn. Howard Hughes Research Labs, Whitehead Institute and Dept, ol Biology. MIT. Cambridge, MA In 1959, Charles Ford, Pat Jacobs, and their colleagues demonstrated that the sex of a developing human embryo is determined byithe presence or absence of the Y chromosome. At the sam e time, Turner syndrome was shown to be associated with a 45.X karyotype. We and others have since observed occasional Turner lem ales with a 46,XY karyotype and deletions of the sex­ determining region ol the Y chromosome. T hese observations suggested that the Turner phenotype might be the result of monosomy tor a gen e or gen es common to the X and Y chromosomes. We set out to explore both the molecular basis ot sex determination and the etiology ot Turner syndrome via molecular genetic studies of the Y chromosome. Among mammalian chromosomes, the Y has been relatively inaccessible to traditional genetic study. Most ot the Y chromosome does not participate in meiotic recombination, and Y-linked g e n e s cannot be readily identified, distinguished one from another, or ordered by meiotic linkage analysis. As a result, genetic dissection of the Y chromosome relies heavily upon the study ot translocations and deletions and the actual cloning ot genes. By examining several hundred individuals with partial Y chrom osom es using a battery of 150 Y-specitic hybridization probes or PCR assays, w e constructed a 38-interval deletion map ol the human Y chromosome. By deletion analysis of human XX males and XY females, we found that sex is determined by a small (280-kilobase-pair) portion ol distal Yp constituting le s s than 0.5% of the chromosome. By deletion analysis ot XY females with and without Turner stigmata, we determined that the "Turner gene(s)" are most likely located within the sam e region, though they appear to be distinct from the sex ­ determining gene(s). Close examination of this 280-kb region, implicated in both sex determination and Turner syndrome, has led to identification of three genes: 1) ZFY encodes a zinc-finger protein that probably activates transcription in a sequence-specific manner. (ZFX, on the X chromosome, encodes a closely related zinc-finger protein.) 2) R PS4Y en cod es an isoform of ribosomal protein S4. ( RPS4X , on the X chromosome, en cod es Ihe other isoform.) S4 dosage may play a critical role in Ihe etiology of Turner syndrome. 3) S R Y (discovered by Goodlellow and Lovell-Badge) encodes a putative DNA-binding protein and plays a pivotal role in gonadal sex determination.

2 MOSAIC EXPRESSION OF MAMMALIAN GENES: IMPLICATIONS FOR DEVELOPMENT AND NEOPLASIA Beatrice Mintz, Institute lor Cancer Research. Fox Chase Cancer Center, Philadelphia. PA Many genes, distributed throughout the genome, apparently express themselves differently among the various developmental clones of which a given cell type is comprised. This generalization is based on evidence In the mouse coat, where genes influencing pigment formation provide visible color indicators ol mosaic expression. Patterns representing clonal variation attributable to specific genes are seen in natural genotypes, and in experimental mice whose color has been increased or decreased by an inserted transgene. The most challenging cases are those due to an autosomal homozygous gene, as such cases must arise by ris-acting genetic mechanisms. Examples of spontaneous and experimental variations in gene expression will be described; and a molecular analysis of the basis for mosaicism in a natural genotype will be presented. Consequences of this flexibility for development and neoplasia will be discussed.

4 ANALYSIS OF ACCESSORY MARKER CHROMOSOMES USING STANDARD AND MOLECULAR CYTOGENETIC TECHNIQUES. Lauren S. Jenkins, L. Bros, S. Rosenfeld, R. Bachman, M. Lipson, D. Witt, and J.Mann Kaiser Permanente Medical Center, Northern California Regional Cytogenetic Laboratory, San Jose, CA 95119 We have used a combination of standard stain­ ing techniques (GTW, CBG, AgNOR, QFQ, RFQ) in addition to the technique of fluorescent in situ hybridization (FISH) to examine accessory marker chromosomes. Ten cases were ascertained by either (1) advanced maternal age indicated prenatal diagnosis [6/10] (2) abnormal post-natal phenotype [3/10] or (3) history of multiple spontaneous abortions [1/10]. The utilization of chromosome specifc centromeric probes by the FISH procedure identifidd the origin of the marker chromosome in four cases: Y derived in two cases; isodicentric chromosome 15 in another case; and iso(18p) in the fourth case. This positive identification of the marker chromosome was a critical piece of information necessary for the diagnosis and prognosis of each case. In the remaining cases these same techniques were used to analyze the content of heterochromatin and euchromatin of these markers. In atleast one case, the marker displayed a single discrete C-band and was negative for R-bands. Whereas, when the no n ­ chromosome specific centromeric probe was used via FISH, the entire marker displayed a fluores­ cent signal. Therefore, the question arises as to which technique gives a more accurate assesment regarding the composition of hetero­ chromatin within marker chromosomes.

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Abstracts - 30th Annual American Cytogenetics Conference

LOCALIZATION OF COSM1DS TO CHROMOSOME 22 BY FLUORESENCE Beatrice S ellin ger1, Yvonne O. Tatsum ura*. Meretc Rasmussen^, Henrik Vissing^. Beverly S. Em anuel*, and Marcia L. Budarf1. 'The Children's Hospital of Philadelphia. Phila.. PA; 2Mt. Sinai Medical Center. NY. NY


Chromosome 22 is next to the smallest of the human autosomes comprising approximately 60 megahases of DNA Despite its relatively small size, numerous rearrangements of this chromosome have been associated with malignant diseases and developmental abnormalities. A long-range molecular map o f this chromosome would allow the more precise localization of the various breakpoints o f these rearrangements. Toward this goal we have constructed a genomic DNA library that allows the isolation o f DNA clones that are directly adjacent to the Notl sites. Notl was chosen because it is a restriction enzyme that digests infrequently in the human genome. Eighty-four cosmids were isolated by screening a library made from Notl digested DNA from a hybrid cell line with chromosome 22 as the only visible human material. In order to independently confirm that the cosm ids were chromosome 22 specific, we mapped the cosmids using nonisotopic in situ hybridization. The cosmids were labeled by biotin incorporation. Avidin-FITC was used for detection, signals were amplified twice and propidium iodide was used for counlcrstaining. To date, 25 cosmids were individually hybridized to normal human metaphascs. The analysis showed that 50 percent of the probes localized to chromosome 22 and the rest were seen on cither C-group or E-group chromosomes. Thus, it appears that the somatic cell hybrid used in the construction of this cosm id library contains fragments o f human chromosomes which arc not derived from chromosome 22. Fluorescence in situ hybridization is a rapid method to determine which cosmids will be useful for further analysis In addition, we are able to sublocalizc the cosmids to the proximal, middle or distal long arm of chromosome 22.

7 USE OF CHROMOSOME SPECIFIC FLUORESCENT PROBES IN CLINICAL CYTOGENETIC STUDIES. Gary Hicks, Mark Law, Eric Christensen, Jack Spurbeck, and S. M. Jalal. Cytogenetics Laboratory, Mayo Clinic, Rochester, MN Fluorescent labelled DNA probes for whole chromosome paints, or-satellite, satellite-ill based heterochromatic regions of 9 and Y, 13-satellite, and the telomere provide a new dimension to chromosome analysis beyond the domains o f chromosome banding. Currently we are using whole chromosome paints for 1-10, 12, and X; a-satellite probes for 1-4. 7, 8, 10-13, 16-18, 20, 21, X, and Y; B-satellite probes for acrocentric chromosomes; heterochromatic probes for chromosomes 9 and Y; and the telomeric probe. These probes have been useful to identify markers, structurally altered chromosomes, and specific chromosomes in interphase. We use these probes on the average o f about seven cases a month. The processing averages 8 minutes for in situ hybridization preparation and 32.6 minutes for post hybridization washes and analysts over a two-day period. Five cases are presented where the new technique has been helpful. Case one was a dysmorphic 15-year-old male. High resolution banded karyotype was 46,XY,-5,+der(5)t(5;?8)(pl5.3;?21). It was suspected that 8p21-pter was translocated to 5pl5.3. This was confirmed by a chromosome 8 paint probe. Case two was an amniotic fluid from a 19-year-old with an elevated AFP. The GTL-banded fetal karyotype was 46,XX,15p-. The entire short arm appeared to be missing. Since the short arm was positive for the hexanucleotide telomeric repeats probe it supported our hypothesis that this was a normal variant. Case three was an 8-month-old male with dysmorphic features. His GTL-banded karyotype was 4 7,XY, + mar. The marker was suspected by G- and Q-banding to be an i(18p). This was confirmed by an a-satellite probe for chromosome 18. Case four was an amniocentesis collected because o f an abnormal ultrasound examination o f the fetus. The fetal GTL-banded karyotype was 46,X Y ,-l,+ der(l)l(l;7)(p 36.3;q22). By sequential G-banding and use o f a paint probe for chromosome 7 the breakpoints were assigned specifically to lp36.3 and 7q22. In cases of hydatidiform moles, triploidy has been detected by use of a combination of a-satellite probes for chromosomes 18, X, and Y from interphase nuclei.

6 THE USE OF FLUORESCENCE IN-SITU HYBRIDIZATION IN THE CHARACTERIZATION OF CYTOGENETIC ABNORMALITIES. Arthur R. Brothman, Ankita M. Patel, Patrick D. Storto, Phillip F. Chance. Eastern Virginia Medical School, Norfolk, and The University of Utah School of Medicine, Salt Lake City. The recent availability of numerous chromosome-specific DNA probes has opened several new avenues of testing for the characterization of cytogenetic abnormalities. While the use of these probes for clinical diagnosis has not yet been federally approved, the present technology warrants their use in confirmation and clarification of certain chromosomal rearrangements. We have used biotinylated probes for both alpha satellite pericentromeric sequences from Oncor and whole chromosome paint (WCP) DNA libraries from Imagenetics in studies to confirm the suspected chromosomal origin of marker chromosomes. While Gand other conventional banding analysis often gives sufficient data, in-situ studies offer support for those data, and in our hands has confirmed unbalanced chromosomal complements in less time than it takes for processing parental blood studies. To date we have used this approach in pre-and post-natal studies involving mosaic trisomy 12p, pericentric inversions of chromosome 4 and various translocations or inversions in different cases involving chromosomes 1, 3, 4, 6, 8, 12 or 22. As all chromosomal sequence probes are likely to become available in the near future, the proficiency in using fluorescence insitu hybridization (FISH) techniques should become routine for all clinical cytogenetic laboratories. Supported by a grant from Children's Health Systems, Norfolk, VA.

8 CYTOLOGICAL IDENTIFICATION O FT E L O M E R IC SEQUENCES AT TERMINAL REGIONS OF C H R O M O SO M E DELETIONS THROUGH FLUORESCENT IN SITU HYBRIDIZATIO N (FISH ). N. Rao, R. Hayworth, M.J. Pettenati. Bowman Gray School o f Medicine o f Wake Forest University, W inston-Salem, N C 27157 The existence o f terminal chrom osom e d eletion syndrom es have led to the long-standing dispute as to whether these d eletion s are simple with the broken ends spontaneously "healing" thus acting as a telomere or whether the telom ere is still present as a result o f an interstitial deletion. T o determ ine if telom eres are present at apparently cytogenetic terminal deletions, FISH was perform ed using biotinlabelled centrom ere and telomere probes, eith er sim ultaneously or in succession. This com bination enabled the d eleted chrom osom es to be identified. Chrom osom e painting was done in som e cases to rule out the possibility o f cryptic translocations. T he follow ing cases with apparently terminal deletions were analyzed: d e l(4 )(p l5 .2 ); del(5) (p 13.3); del(6)(p23); del(8)(p23.1); d e l( 1 8 )( p ll) and del(18)(q21.3). Protocols were that provided by the supplier with som e modifications. 20 m etaphases from each sample were analyzed for presence of telom eres at the end o f deleted arm. In nearly 80% o f the cells 1 or 2 distinct telom eres were observed at the d eletion site. These observations implied that either all the d eletions w ere interstitial with intact telom eres or that capping with telom eric sequences had occurred at the d eleted site. There is evid en ce o f the latter occurring in humans, by the existence o f an enzym e telom erase. T his enzyme, an unusual ribonucleoprotein reverse transcriptase, has b een shown to add telom eric repeats de novo onto the non-telom eric ends generated by deletions. Our finding provides direct cytological evidence that telom eres are present at apparently terminal deletions, suggesting that chrom osom al deletions may be interstitial or that broken chrom osom e regions are "healed" by the addition o f telom eric sequ en ces. Based on cytogenetic evidence, we favor the latter to be occurring in some instances. This ensures the deleted chrom osom e from further breakage or fusion with other chrom osom e or shortening during replication thus maintaining its length in successive cell generations.

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Abstracts - 30th Annual American Cytogenetics Conference

A CASE OF DEL iq42.3 DUE TO A DE NOVO NONRECIPROCAL TRANSLOCATION (1;8) CONFIRMED BY FLUORESCENT IN SITU HYBRIDIZATION. J.C. Stout, J.E. Wiley, T. Kushnick. East Carolina University School of Medicine. Chromosomes from a child born with multiple congenital anomalies were studied with high resolution banding and found to have an apparently nonreciprocal translocation (1;8) (q42.3;q22.1). While the 8q22.I-^qter segment was clearly translocated to the distal portion of the long arm of chromosome 1, the lq42.3-^qter segment did not appear to be attached to 8q. In order to further establish whether the translocation was reciprocal or not, fluorescent in situ hybridization (FISH) was performed using whole chromosome paints for chromosomes 1 and 8. As expected, metaphases hybridized with the chromosome 8 paint revealed three fluorescent signals corresponding to the normal 8, the deleted 8, and the translocated 8q22.1-^-qter segment which was attached to the distal lq. Conversely, raetaphases hybridized with the chromosome 1 paint yielded only two fluorescent signals which were found on the normal chromosome 1 and part of the derivative chromosome 1. The lack of hybridization of the chromosome 1 paint to distal 8q further confirmed the nonreciprocal state of the translocation. The proband's phenotypic abnormalities were consistent with deletion lq syndrome which involves the same breakpoint on chromosome 1.

11 SUPERNUMERARY i(5P) IN A CHILD AND A DIFFERENT MARKER CHROMOSOME PRESENT IN HER MOTHER. Wayne Stanley, Kenneth Rosenbaum, Grace Devine, Tara Ellingham and Bridget Murphy. Departments of Laboratory Medicine and Medical Genetics, Children’s National Medical Center, Washington, D.G 20010. Trisomy 5p syndrome is associated with a phenotype consisting of hypotonia, macrocephaly, seizures, psychomotor retardation, low-set or dysplastic cars, and slanted palpebral fissures. In most cases, this duplication results from familial translocations but there are three reports o f 5p trisomy due to isochromosome 5p formation. To our knowledge, however, there are no reported cases of tetrasomy 5p. We found in a 5-year-old slightly dysmorphic girl with seizures, psychomotor retardation, and diffuse areas of hyperpigmentation a supernumerary marker chromosome in fibroblasts (60% of cells). By GTG-banding, this marker appeared to be an i(5p) which was confirmed by fluorescence in situ hybridization (FISH) using an a-satellite probe specific for the ccntromeric region of chromosomes 1, 5, and 19. The prevalence o f this marker in the population may be more common but undetected because of cither limited tissue distribution and/or misdiagnosis due to its close resemblance to the i(12p) described frequently in patients with pigmentary abnormalities. This patient's mother had a morphologically different supernumerary marker chromosome in her lymphocytes. This small satellited marker was mostly heterchromatic and did not hybridize with probes to chromosomes 13/21, 1/5/19, or 15. The presence of different marker chromosomes in this family could be coincidental or possibly indicate familial genomic instability leading to supernumerary marker formation.

10 AM APPARENTLY BENIGN EUCHROHATIC DUPLICATION IN CHROMOSOME 9: CYTOGENETIC AND H3LECULAR CHARACTERIZATION. Lawrence CharlT.y, Gail Stetten, Carla Rosenberg, and Judith Staaberg. Union Memorial Hospital and Johns Hopkins Univ. School of Hedicine, Baltimore, MD. Chromosome 9 is well known for its heterochromatic region, which has common variations in size. Inversions of this region are also frequently found and are considered to be normal variants. Here we report an unusual chromosome 9 which appears to have a duplication of a euchromatic band just above the centromere. This chromosome was found in a fetus and the phenotypically normal mother. The patient was a 26-year-old woman referred for amniocentesis due to an elevated serum alpha-fetoprotein level. G banding showed a metacentric chromosome 9; however, the heterochromatic region appeared to be entirely below the centromere. C banding confirmed that there was no heterochromatin in the short arm. An unbalanced translocation was suspected; parental blood studies revealed that the mother possessed the same metacentric chromosome 9 and no other apparent aberrations. Fluorescent in situ hybridization studies with a chromosome 9-specific library were then performed. In both fetus and mother the entire #9 chromosome fluoresced; no other chromosome had any fluorescence. This indicated that the extra material above the centromere was of chromosome 9 origin; furthermore, no chromosome 9 material had been translocated to any other chromosome. Host likely, the extra material is a duplication of band 9pl2. Although the duplicated region is euchromatic, a clinically normal parent had the same chromosome and the family was counseled that the fetus is likely, though not certain, to be unaffected. Sutherland and Eyre found an apparently similar case in one family which they studied by G and C banding (Clin. Genet. 19:331,1981; case 2). The pregnancy is continuing and follow-up studies on the outcome are pending.

12 CYTOGENETIC AND MOLECULAR ANALYSIS OF NON-DISJUNCTION IN TRISOMY 21. *B.J.Lorber.^M.Grantham,'ll.Peters,aH.F.Willard, and*T.Hassold. *Emory University, Atlanta, GA;aStanford University, Stanford, CA. Over the past 20 years, cytogenetic heteromorphisms have been used extensively to study the origin of trisomy 21. From these studies, it has been estimated that as much as 20-30Z of trisomy 21 is paternally derived. However, the results of recent DNA marker studies of trisomy 21 indicate a paternal non-disjunction rate of only 5Z. From this, it has been suggested that the cytogenetic approach has an un­ acceptably high error rate, and should be abandoned. Despite this, we think that cytogenetic heteromorphism evaluation may still be valuable, as there are no highly polymorphic DNA markers at the centromere of chromosome 21. Therefore, the meiotic stage of origin cannot be assessed by molecular techniques. Because of this, we have been in­ terested in improving the reliability of the cytogenetic approach by adopting a more conservative scoring system to reassess classical cytogenetic heteromorphisms and have in­ vestigated a molecular cytogenetic approach using fluor­ escent in situ hybridization to beta-satellite DNA sequences located in the short arms of the acrocentrics (beta-FISH). To date, we have studied 56 trisomy 21 families with DNA markers, Q-banding, and/or beta-FISH. Our results indi­ cate that Q-banding and beta-FISH may recognize different heteromorphisms, but overall the frequency of heterozygosity is about 25Z by both techniques. Therefore, beta-FISH is a useful adjunct to classical cytogenetic techniques for stud­ ies of non-disjunction in trisomy 21. There was 100Z concor­ dance between the cytogenetic results and the DNA marker studies in determining parental origin, indicating that ju­ dicious use of chromosome heteromorphisms may be useful in parental origin determinations. However, there was a sur­ prising level of discrepancy between the cytogenetic and molecular techniques in determining the meiotic stage of non-disjunction which could be explained by previously un­ recognized crossing-over between the non-disjoined chromo­ somes 21.

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Abstracts - 30th Annual American Cytogenetics Conference

X-INACTIVATION PATTERNS IN MULTIPLE TISSUES OF THREE C A SE S OF A PPA RENTLY BALANCED X /A U T O SO M E TRANSLOCATIONS WITH ABNORMAL PHENOTYPES. JL Zackowski', ES Cantii2, DJ Driscoll1, H Ostrer3, RT Zori1, BA Gray'. 1 R.C. Philips Unit, University o f Florida Health Science Center, Gainesville; 2 Medical University o f South Carolina, Charleston; 3 New York University Medical Center, New York. Cytogenetic late replication studies o f apparently balanced X/autosome translocations, utilizing peripheral blood lymphocytes, have shown a preferential pattern o f normal X chromosome (X J inactivation in a majority o f cases reported, including some cases where the phenotype expressed is abnormal. In a small number o f such abnormal cases reported in the literature, inactivation o f the translocation X chromosome (XJ has been cytogenetically observed in other tissue types subsequently studied. Such observations directly implicate an X-inactivation pattem/phenotype relationship, and suggest diagnostic and/or prognostic value for late replication studies in more than one tissue type. We report three cases of apparently balanced X/autosome translocations with abnormal phenotypes: 46,X ,t(X ;9)(p22.1;q32)mat (diagnosed prenatally), 46,X,t(X;9)(p22;q21), and 46,X,t(X;4)(q24;q25). In all three cases, Xinactivation patterns were studied in more than one tissue type (lymphocytes and fibroblasts or amniocytes) in an attempt to elucidate the etiology o f the abnormal phenotypes. In the first case, all amniocytes examined demonstrated exclusive inactivation o f X„, while among lymphocytes, some cells showed X„, and other cells X,, to be late replicating. However, in the remaining two cases, exclusive inactivation o f X„ was observed in both tissue types examined. These cases are of interest because: (1) they provide further documentation o f patterns o f Xinactivation in cases of balanced X/autosome translocations with abnormal phenotypes, and (2) they demonstrate that cytogenetic studies of Xinactivation patterns, even in two tissue types, are not necessarily of diagnostic or prognostic value in cases o f balanced X/autosome translocations. 15 CHROMOSOME BREAKAGE IN HOMAN SPERM. A.M. Estop1-*, T. Levinson1-*, and S. Hunne1-*. (1) West Penn Hospital, Pittsburgh and (2) University of Pittsburgh, Pittsburgh, PA Cytogenetic studies of human sperm allow us to evaluate the extent of chromosome damage in a healthy man. The incidence of sperm chromosome structural abnormalities in individual men is highly variable and it may range from 0 to 25%, revealing that this cell type is prone to de novo structural abnormalities. The characterization of these structural abnormalities and their breakpoints will allow us to (1) determine which chromosomes are more frequently involved in sperm structural abnormalities and whether repeated specific breakpoints are found in sperm cells; (2) establish a correlation between the breakpoints of sperm chromosome lesions and fragile sites, if any; and (3) speculate about the origin of these structural aberrations in relation to the process of sperm formation. Seven men with normal constitutional karyotypes plus 4 translocation carriers were studied by allowing the fusion of sperm cells with hamster oocytes. The sperm pronuclei then developed into haploid chromosomes. 1,233 sperm complements were available for cytogenetic analysis. 109 break events corresponding to 103 structural abnormalities were found. The precise location of 80 chromosome break point b was determined. A number of chromosome break points were observed more than once. Most of the chromosome structural abnormalities found were unrejoined events such as chromosome breaks, fragments and deletions. Twenty-four of 80 break points of chromosome lesions (30t) fall in chromosome fragile sites. This frequency tells us that break events in sperm do not occur preferentially in fragile sites.


M J. Pettenali and N. Rao. Bowman Gray School o f Medicine o f Wake Forest University, Winston-Salem S C We present a review of 431 paracentric inversions (PAI) [312 literature; 119 submitted] to better understand its’ occurrence, nature and clinical relevance. PAI were present in all chromosomes. Chromosome arms 18p, 19q, 20q and Yp were devoid o f PAI. The most com m on inversions occurred in chrom osom es 11, 6, 7, 1, 5, and 3, respectively. Less than 1% were in chrom osom es 19, 20, 22, Y, 21, 16, 18, 4, and 17. Significantly more than expected PAI were noted in chrom osom es 1, 6, 7, 11 and 12 and in chrom osom e arms 6p, 7q, llq , 12q and Xp. Significantly less were observed in chrom osom e arms 2p, 2q and 4q. Most frequently encountered PAI were: Ilq21q23, 7ql lq22, 6pl2p23, 6pl2p25 and 3pl3p25, respectively. Inheritance of PAI were as follows: familial-65.7%; denovo8.8%; unknown-25.5%. Reasons of ascertainm ent included: systematic -54.5%; M R/CA-22.3% ; SA B -11.6%; syndrome associated-3% ; infertility -2.1%; unknown-6.5%. The average inverted segmental lengths were unrelated to ascertainment. A high incidence o f aneuploidy (n = 12) was noted among PAI carriers implying a possible interchromosomal effect. There did not appear to be an association o f ascertainment with a specific PAI. However, significant relationships were noted for associations between M R /C A and chrom osom e 1; SAB and chrom osom es 13, 1 and 14; clinical features o f Turner syndrome and the X chromosome. There were 15 instances of recombinant chromosomes: 2 dicentric and 13 monocentric (7 dup/5 d e l / 1 dup-del). These outcomes suggest that PAI recombination events result m ore often in monocentric chrom osom es rather than the expected dicentric formation. There were no common factor suggesting a tendency towards recombination. The incidence o f recombination is estimated at 1-2%. li t i s review demonstrates that PAI are more common than suggested by previous reviews. Despite biases o f ascertainment in some instances, there appears to be associated risks with PAI that requires further examination.


METAPHASE PREPARATION FROM CULTURES ARRESTED IN G2 BY TOPOISOMERASE I N H I B I T I O N . J o h n F. S to n e , S outhw est Biom edical R esearch I n s t i t u t e , S c o t t s d a l e , AZ S u b strate-attach ed c e ll c u ltu re s are d i f f i c u l t to sy n ch ro n ize w ith th e te c h n iq u e s commonly u s e d f o r ly m p h o c y te s y n c h r o n i z a t i o n . We h a v e e x p l o r e d t h e i n h i b i t i o n o f to p o i s o m e r a s e I I ( t o p l l ) a s a means of a r r e s t i n g c e l l s i n G2 , f o l l o w e d b y r e l e a s e of th e b lo c k and c o l l e c t i o n of m e ta p h a se s . U sing t h e i n h i b i t o r H o ech st 33342 (H 33342), we h a v e f o u n d a 2 1 / 2 - 3 f o l d I n c r e a s e i n t h e m i t o t i c i n d e x (MI) of t r e a t e d c u l t u r e s . In a d d i t i o n , t h e chromosomes a p p e a r e d lo n g e r th a n controls. C ontinuous exposure to th e b lo c k i n g a g e n t r e s u l t e d in an a p p r o x i m a t e ly e q u a l MI a s w h e n c e l l s w e r e r e l e a s e d f r o m t h e block p r i o r to c y to g e n e tic p r e p a r a ti o n . T his s u g g e s t s t h a t H33342 b o t h r e t a r d s t h e c e l l s ' p a s s a g e t h r o u g h G2 a n d i n h i b i t s c h r o m o s o m e condensation. S im ila r r e s u l t s were o b ta in e d from f i b r o b l a s t s , a m n io c y te s , c h o r i o n i c v i l l u s c e l l s , c e l l l i n e s and p rim a ry s o l i d tum ors. We a r e c u r r e n t l y t e s t i n g t h e e f f e c t of E to p o s i d e , w hich i n h i b i t s t o p l l by a m echanism d i f f e r e n t from H33342. R e su lts of t h e s e e x p e r i m e n t s w i l l be d i s c u s s e d . Downloaded by: Karolinska Institutet, University Library - 1/17/2019 7:15:12 AM


Abstracts - 30th Annual American Cytogenetics Conference

prenatal Cytogenetic Diagnosis of the Fragile X Chromosome: Feasibility and Speed of In Situ Clonal Method in Amniotic Fluid Cell Tissue Culture. Lawrence R. Shapiro, Patrick L. Wilmot, and Lynn E. Andree. Departments of Pediatrics and Pathology, Westchester County Medical Center and New York Medical College, Valhalla and Regional Medical Genetics Laboratory, Thiells, New York, USA. Until now, prenatal diagnosis of the fragile [fra(X)] chromosome in amniotic fluid has necessi­ tated the use of the flask method of tissue culture with extended tissue culture periods of 3-4 weeks. The use of the in situ clonal method for routine prenatal cytogenetic diagnosis of amniotic fluid cells has shortened tissue culture time. Simultan­ eous use of the flask method and in situ clonal method of tissue culture for prenatal fra(X) chromo­ some analysis has been employed by us, and we have found 2 cytogenetically fra(X) positive cases in amniotic fluid cells cultured by both methods. The first case involved a .125 a priori risk for a female fetus which was 4% fra(X) positive in the flask method which required 4*s weeks to report. Utilizing the in situ method, it was found in 2*s weeks that 1/8 clones were cytogenetically fra(X) positive, and 1/19 cells were fra(X) positive in that clone. The second case was a male fetus with an a priori risk of .5 which was 13.3* fra(X) positive in the flask method in 5 weeks. Usinq the in situ method, 2/7 clones were cytogenetically fra(X) positive in 2% weeks. 1/16 cells were cytogeneti­ cally fra(X) positive in 1 clone and 1/11 cells was fra(X) positive in the second clone. While all cells in a clone are genetically identical, it is reasonable for only 1/19, 1/16, or 1/11 cells in a clone to be cytogenetically fra(X) positive because the induction of the fra(X) chromosome is likely to be dependent on the metabolic state of each individual cell.

19 THE USE OF FLUORESCENCE IN SITU HYBRIDIZATION TO DETECT SPECIFIC CHROMOSOMES IN UNCULTURED FETAL CELLS. G. Prabhakar. K.J. Blakemore, V .L. Jaswaney and G. Sietten. The Johns Hopkins University School o f Medicine, Baltimore, MD. Fluorescence in situ hybridization (FISH) with centromeric DNA probes is emerging as a rapid, chromosome-specific cytogenetic screening tool. This study describes a reliable method for preparation o f cells for FISH directly from uncultured chorionic villi and amniotic fluid. In this study we compare interphasc cell nuclei from these two sources. Uncultured chorionic villi were prepared by the method o f Simoni el a l., (1983) omiting the 24 hr incubation, Colcemid exposure and slide heating. Amniocytes were prepared by direct hypotonic treatment and methanol-acetic acid fixation. For fluorescence in situ hybridization (Pinkel el a l., 1986) 20ng o f probe was added to the hybridization mixture (60% formamide/ 2XSSC). Probe and target DNA were simultaneously denatured on slides al 75-80 C for 5 min. After overnight incubation at 42 C, the slides were washed at high stringency. The probes were detected with FITC conjugated avidin after one round o f amplification. First trimester chorionic villus samples were studied for the following indications: sex determination, mosaicism for an autosomal trisomy, possible triploidy and identification o f a small marker chromosome. Uncultured amniocyte samples were studied to determine fetal sex or trisomy status. Compared to uncultured chorionic villus cells, amniocytes displayed a marked heterogeneity in cell morphology. Only 10-20% o f the amniocyte nuclei could be used for FISH analysis whereas 95-100% of the dispersed trophoblastic chorionic nuclei were usable. Both villus and amniotic fluid cells were examined at several different gestational ages. Chorionic villus direct preparations were superior to amniocytes both in terms o f % cells with a positive signal and signal clarity. However, diagnosis was possible in all cases and was later confirmed by chromosome analysis.

18 THE NUMBER OF CELLS ANALYZED TO DETECT MOSAICISM. Julie Zenger-Hain, Denise Kramer, Flonia Myrick, i Daniel L. Van Dyke, Medical Genetics and Birth Defects Center, Henry Ford Hospital, Detroit, Michigan. Perhaps the single most important reason to examine multiple cells in karyotype analysis is to detect mosaicism. The current CAP recommendation is to analyze 20 cells in most situations. We conducted a retrospective study to examine how many analyzed cells were necessary to suspect and confirm mosaicism in peripheral blood and amniotic fluid samples. Our protocol for number of cells to analyze includes 10-30 cells depending on tissue and reason for karyotype. Between 1/1/78 and 5/31/91, 10,690 peripheral blood samples were analyzed and 1.03% (n=110) were mosaic. Mosaicism was suspected when the first non-modal cell was identified and mosaicism was confirmed with the second non-modal cell. Mosaicism was suspected in 90% of the samples by the 7th cell and 90% were confirmed by the 20th cell, with no difference between the autosomal and sex chromosome mosaics. Between 1/1/78 and 12/31/87, 10,024 amniotic fluid samples were received and harvested in suspension and 0.19% (n=19) were mosaic. 90% of the mosaics were suspected and confirmed by the 6th and 10th cells, respectively. A total of 7,365 amniotic fluid samples were received and harvested in situ between 1/1/88 and 5/31/91, and 0.3% (n=21) were mosaic. 90% of the mosaics were suspected by the 7th cell, and 90% were confirmed by the 13th cell. The cell number at which mosaicism was suspected and confirmed was similar between the harvest methods, but the proportion of mosaic samples was slightly different between the harvest methods (0.19 vs. 0.3%) (p< 0.20), and a lower median rate of mosaicism was found by in situ (28% of cells abnormal) than by suspension (48% of cells abnormal) harvests, so it is plausible that the in situ method is more effective in detection of mosaics. Validation studies are being conducted to develop a protocol for number of cells to analyze to detect clinically significant mosaicism based on tissue, culture type, and reason for karyotype.

20 HYBRIDIZATION OF TELOMERIC DNA TO INTERSTITIAL CHROMOSOMAL SITES: CORRELATION WITH FRAGILE SITES AND BREAKPOINTS OF CHROMOSOMAL REARRANGEMENTS. S. Adekunle. A. Milunsky and H.E. Wyandt, Center for Human Genetics, Boston University School of Medicine. Boston MA 02118 Fluorescent in situ hybridization (FISH) of the telomeric sequence, (TTACGC)n , has been applied to chromosome prepara­ tions from a variety of human cell lines including transloca­ tions, deletions, duplications, rings, inversions and other structural abnormalities. Chromosome preparations which were G-banded prior to or following in situ hybridization predict­ ably showed hybridization to the ends of chromosome arms. In addition, however, less intensely fluorescent interstitial sites of hybridization were seen, following high and low stringency post-hybridization washing. Of 21 cell lines studied, all showed 2-7 interstitial sites per metaphase spread with high stringency washing, while two of the lines for which hybridization was repeated with low stringency washing, showed 20 or more sites per metaphase spread. All of the interstitial sites correspond to some rare fragile sites or to common fragile sites inducible by various agents. Two interstitial sites observed at Xql3 and 3q23 have not been reported as fragile sites. The most frequent interstitial sites correspond to the most frequent fragile sites. Also, some of the interstitial sites corresponded to breakpoints of the rearrangements included in the study, especially rings, inversions, and translocations. The implications of such interstitial sites are discussed. The conservation of fragile sites across species (Stone et al, 1991), the similarity of the mode of induction, the clustering of spontaneous breakage and recombination at fragile sices all fit the model of unstable interstitial telomeres studied in yeast (Murray et al. 1988). We, therefore, propose that inter­ stitial telomere-like sequences generally may be the predispos­ ing source of fragile sites, and may have had an important role in chromosome evolution. References: Murray AW et al (1988). Mol Cell Biol, 8:4642-4650. Stone DM et al (1991). Am J Med Genet, 40:214-222.

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Abstracts - 30th Annual American Cytogenetics Conference

FRAGILE X CYTOGENETIC GUIDELINES REVIEWED: RELEVANCE TO FRA(X) MOLECULAR GENETIC STUDIES AND TO APPROPRIATENESS OF CHROMOSOME STUDIES IN ALL CASES OF DEVELOPMENTAL DELAY. A DISCUSSION. PB Jacky and EC Jenkins. Department of Cytogenetics/Molecular Genetics, Kaiser Permanente NW, Portland, OR, and Department of Cytogenetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY. Recent developments in the specific DNA molecular diagnosis of fra(X) syndrome and the lack of reliability of certain aspects of establishing the diagnosis cytogenetically, particularly in at risk carrier females and unaffected transmitting males, have raised questions about the continued use of chromosome studies in establishing the fra(X) diagnosis in individuals and in families. Specific cytogenetic guidelines for fra(X) studies have been generally adopted (Jacky et al. Am J Med Genet. 38:400-403, 1991), and an ad hoc committee recently convened at the Fifth X-Linked Mental Retardation Workshop in Strasbourg in August, '91, reviewed those guidelines, and developed some concensus as to the continued appropriateness of chromosome studies in lieu of recent molecular diagnostic developments (Webb, J Med Genet 28:814-817, 1991). The committee recommendations were: 1. that all previously adopted fra(X) cytogenetic guidelines remain functionally appropriate and useful in the chromosomal diagnosis of this disorder, 2. that fra(X) chromosome studies were appropriate in the evaluation of all individuals, male or female, with significant mental retardation or developmental delay, independent of etiology, clinical phenotype, or family history, and 3. that chromosome studies remain the primary diagnostic test in identifying the fra(X). Once the fra(X) diagnosis is established, a molecular evaluation of immediate and extended family members is likely appropriate. The guideline revisions were considered appropriate given the general reliability of fra(X) chromosome studies in affected males and females, and the high prevalence of non-fra(X) chromosome abnormality in this patient referral population.

23 CHROMOSOMAL RELATIONSHIPS BETWEEN PEROMYSCUS OREAS AND L i s a R. S mi t h and I r a F. Greenbaum, D e p a r t m e n t o f B i o l o g y , T e x a s A6iM U n i v e r s i t y , C o l l e g e S t a t i o n , TX 77843.


Peromyscus from n o r t h o f t h e C o l u mb i a R i v e r and w e s t o f t h e C a s c a d e M o u n t ai n s a r e now r e c o g n i z e d t o c o n s t i t i t u e 2 s p e c i e s , P. maniculaCus (austerus) and P. oreas. A l t h o u g h t h e s e mi ce a r e b r o a d l y s y m p a t r i c and m o r p h o lo g i c a ll y s i m i l a r t h e i r k a r y o t y p e s (2n-48; FN- 72- 78 a n d FN-80-92, r e s p e c t i v e l y ) a r e non­ o v e r l a p p i n g , a n d chromosomal i n t e r m e d i a t e s h a v e n o t been observed. T h e r e a r e , h o we v e r , no d a t a r e l a t i v e t o t h e p o t e n t i a l chromosomal i n t e r a c t i o n b e t w e e n P. o r e a s a n d P. m. artemisiae, t h e m o r p h o l o g i c a l l y d i s t i n c t h i g h FN ( - 8 2 - 8 6 ) d e e r mouse w h i c h o c c u r s j u s t t o t h e e a s t o f t h e r a n g e o f P. oreas. We c o l l e c t e d d e e r mi ce f r om a known p o p u l a t i o n o f P. o r e a s a n d e a s t i n t o t h e r a n g e o f P . m. artemisiae. W i t h i n t h i s r a n g e we o b t a i n e d mi c e o f b o t h m o r p h o l o g i c a l for ms w i t h i n t h e same t r a p l i n e s . G— a n d C- b a n d d a t a h a v e e x t e n d e d t h e known r a n g e o f c hr omos oma l v a r i a t i o n i n P . m. artemisiae ( F N - 8 0 - 8 6 ) a n d i n d i c a t e t h a t t h e two f o r ms d i f f e r f o r chromosomal a r r a n g e m e n t s due t o p e r i c e n t r i c i n v e r s i o n s an d t h e p r e s e n c e o r a b s e n c e o f h e t e r o c h r o m a t i c s h o r t a r ms . The chromosomal d a t a p r o v i d e no e v i d e n c e f o r h y b r i d i z a t i o n b e t w e e n t h e s e two d e e r m i c e .

22 ANEUPLOIDY SECONDART TO GENE TARGETING IN EMBRYONIC STEM CELLS. J. T. M a s c a r e l l o , B. R oller and 0. Sm ithies. G enetic S ervices, C h i l d r e n ’ s H o s p i t a l , 8001 F r o s t S t . , San D i e g o , CA 92123 and D ept, of P athology, Univ. of N o r t h C a r o l i n a , C h a p e l H i l l , NC 27599 T a r g e t e d r e c o m b i n a t i o n was u s e d t o " k n o c k o u t ” t h e f u n c t i o n o f one copy o f t h e - m i c r o g 1o b u l i n gene in mouse em bryonic stem (ES) cells. C h im e ra s g e n e r a t e d from t h e r e c o m b i n a n t c e l l s were then used to produce mice who were d eficien t in P ,-m icroglobulin (hom ozygotes). Some, b u t n o t a l l , o f t h e h e t e r o z y g o u s m a l e s were i n f e r t i l e . Chromosome a n a l y s i s o f t h e s e m a l e s was u n d e r t a k e n a s p a r t o f an a t t e m p t t o exclude "triv ial" explanations for their in fertility . Of t h e f o u r m a l e s s t u d i e d , t h r e e h a d a n e u p l o i d k a r y o t y p e s a n d o n e was m o s a i c w ith a m inority cell type that was also aneuploid. The e x t r a c h ro m o so m e i n t h e m o s a i c mouse c o u ld n o t be i d e n t i f i e d b u t the e x tra chrorasome in t h e n o n -m o s a ic m ice w as. One h a d a n e x t r a X. On e h a d a n e x t r a Y, a n d t h e l a s t h a d an e x t r a chrom osom e 3. W h i l e t h e ES l i n e u s e d in t h i s e x p e r i m e n t d o e s h a v e an u n u s u a l l y high p ro p o r tio n of h y p e rd ip lo id c e l l s , it is s t i l l not c l e a r w hether the fr e q u e n t p ro d u c tio n of aneuploid mice in the experim ent was a f u n c t io n o f i n s t a b i l i t y in th e p a r t i c u l a r l i n e , in stab ility i n ES c e l l s (in g en eral), o r an i n s t a b i l i t y t h a t was s e c o n d a r y t o t h e c h i m e r i c c o n d itio n of the hom ozygote’ s p a r e n ts . 24

CHROMOSOMAL FRAGILE SITES IN IT IF. DEER MOUSE: DISTRIBUTION, VARIATION, AND POTENTIAL ROLE IN CHROMOSOMAL EVOLUTION. Bryant F. McAllister and Ira F. Greenbaum. Department o f B iology, Texas A&M University, College Station, TX 77843. This research examined the occurrence o f chromosomal polym orphism s and the chromosomal location of aphidicolin-induced fragile sites in wildcaught deer mice, Peromyscus maniculatus. The analysis o f chromosomal breaks to determine fragile-site location was performed separately for each individual. This allowed the identification of interindividual variation in fragile-site constitution and expression. Under certain assumptions, individual fragile-site genotypes were determined and allele frequencies were estim ated. For all fragile sites the alleles follow ed a Mendelian model where the allelic distributions fit Hardy-W einberg expectations. In addition, fragile sites observed at high frequencies within populations were shared between two geographically separated sam ples. Inierpopulational differences occurred at fragile sites observed in small numbers of individuals within sam ples. Three pericentric inversion polym orphism s are maintained within these populations. Location o f fragile sites at two o f the three inversion regions indicates a potential role of fragile sites in chromosomal rearrangement. Further, these data suggest that pericentric inversion polymorphisms within populations o f P e r o m y s c u s are due to increased mutation rates at fragile sites.

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Abstracts - 30th Annual American Cytogenetics Conference

KARYOTYPES OF TWO PORCINE RENAL CELL LINES. Janet M. Cowan, Dept Pediatrics, Eastern Virginia Medical School, Norfolk, VA 232501 LLC-PK/C14 is a porcine renal line capable of differentiating into a polarized monolayer at confluence, with characteristics of cells of the proximal tubule of the kidney. It has been widely used to study the distribution and functions of renal transporters. In order to determine the location of the antiporter, which contributes to the regulation of intracellular pH, cell volume and transcellular sodtum ion transport in the kidney, a mutant line (PKE20) was derived from LLC-PK/C14. PKE20 has a 3 - 5 fold increase in antiporter activity. Since the increased antiporter activity might be the result of gene amplification both parental and mutant cell lines were karyotyped to determine if there were any homogeneously staining regions (HSRs) or double minutes (DMs) that could the sites of gene amplification. No HSRs or DMs were observed, but the karyotype of the mutant line differed from that of the parental line. The parental line had an abnormal karyotype and included an apparent Robertsonian fusion of chromosomes 15 and 17. The mutant line appeared to have loss this chromosome, and gained an isochromosome 17, resulting in trisomy. These results suggest that chromosome 17 may be the location of the gene(s) that contribute to antiporter activity.


CONSISTENT NUMERICAL CHROMOSOME ABERRATIONS IN CONGENITAL FIBROSARCOMA. Susanne M. Gollin, Suguna Sankary, Eugene Wiener, William Robichaux, William P. Swaney, Patrick S. Malone, and Paul S. Dickman. Departments of Human Genetics (S.S., S.M.G.), Surgery (E.W.), and Pathology (P.S.D., W.R.), at the University of Pittsburgh, Children’s Hospital of Pittsburgh, and the Pittsburgh Cancer Institute, Pittsburgh, P A Congenital (infantile) fibrosarcoma is a low grade malignant spindle cell neoplasm of soft tissue composed o f fibroblasts and myofibroblasts. In our case of a congenital fibrosarcoma of the volar forearm from a five month old boy, extensive immunostaining of tumor cells for actin and vimentin indicates that myofibroblasts were the dominant cell population. Cytogenetic analysis of 20 trypsinGiemsa banded metaphase cells from passage 2 cell cultures (named PCI:SG530) showed 8 cells with a 46,XY, apparently normal male chromosome complement and 12 cells with a 4 9 ,X Y ,+ 1 1 ,+ 17. + 20 chromosome pattern. No structural aberrations were observed. Karyotypes of four previously reported cases of congenital fibrosarcoma were (1) 47,XY, + 8 /4 7 ,X Y ,+ 11/48,X Y ,+ 8 ,+ 1 1 / 49,X Y ,+8, +11,+20; (2) 46.X Y/49.XY ,+8, +11, + 20/50,XY, + 8, +11, + 17,+20; (3) 49,X,-X,+ 11,+11,+ 17,+ 20; and (4) 48.XY.+ 11,+ 20. Although no single chromosome gain is common to all clones of these five cases of congenital fibrosarcoma, trisomy 11 appears to be the characteristic finding with trisomies 8, 17, and 20 occurring frequently. Growth-related genes on chromosome 11 include insulin, insulin-like growth factor 2, and various oncogenes. In addition, chromosome abnormalities associated with Beckwith-Wiedemann syndrome, an overgrowth syndrome, involve chromosome lip . The role of these genes in the development of congenital fibrosarcoma merits further investigation.

26 CYTOGENETICS AND ORIGINS OF OVARIAN GERM CELL TUMORS L. Hoffner. R. Deka, R.E. Ferrell, A. Chakravarti and U. Surti C ytogenetic and ONA fingerprint analysis w a s performed on three c a s e s of malignant ovarian germ cell tum ors, nam ely, one malignant teratoma with yolk sa c elem en ts, one malignant teratoma with elem en ts of endoderma! sinus tumor, embryonal carcinom a, and choriocarcinom a, and one malignant teratoma with yolk sa c elem en ts and embryonal carcinoma. Each tumor and corresponding normal tissu e w a s karyotyped and scored for centrom eric heterom orphisms to determ ine the m echanism of origin. The karyotypes were 7 9 .XXX, 6 .+ 1. + 3 .+ 3 .+ 8 , + 12 . + 14. + 15. + 17. + 2 0 .+ 2 1 , + 2 2 ; 4 9 ,XX. + 8. + 12. + 22; and 48.X X . + 3. + 14. The first c a s e also contained an i(12)p marker chrom osom e in 2 out of 3 8 cells. The anlysis of centromeric heterom orphisms and DNA fingerprints of h ost and teratoma using the Ml 3 probe revealed that one ca se originated from a germ cell before the first m eiotic division. Normal host tissu e w a s not available in ca se 92, but several centromeric markers were h eterozygous in the tumor, indicating either m eiosis I error or com plete failure of germ cell m eiosis. In the third c a s e the centromeric heterom orphism s that w ere h eterozygous in the host appeared to be h om ozygous for certain ch rom osom es and heterozygous for others in the tumor. T h ese results su g g est its origin is by the fusion of tw o ova. In addition, w e have com pleted cytogen etic analysis on 1 6 7 benign ovarian teratom s. Our c a s e s, in addition to 188 other c a s e s reported in the literature, revealed the following chrom som al abnormalities: 47.X X . + 8 (4 cases); 4 7 ,XX, + 12; 4 7 .XX. + 14; 4 7 .X X .+ 15; 4 7.X X . + 16; 47.XXX; 47 .X X .-1 5. + 2 1 . + mar; 48.X X . + 7. + 12; 48.X X . + 8 . + 8; 69.XXX; 92.X X X X /91.X X X X .-13. Cytogenetic data on malignant ovarian germ cell tumors available to date indicate the p resence of nonrandom numerical abnormalities. In particular, ch rom osom es 3 , 12. and 14 appear to be present in additional cop ies. Chromosomal abnormalities are much more frequent in malignant ovarian germ cell tumors (80% ) a s compared with the benign teratom as (3%).


THE RECURRENT CHROMOSOME CHANGES IN SQUAMOUS CELL CARCINOMA OF THE HEAO AN0 NECK REGION. D.L. Van Dyke, M.J. Horsham, M.J. Benninger, T.E. Carey. Henry Ford Hospital, Oetroit, MI; University of Michigan, Ann Arbor, MI 48109 Our group has characterized the breakpoints, gains, and losses of chromosome material in squamous cell carcinoma (SCC) of the head and neck region from 23 patients. Cell lines were studied in 1/3 of cases, direct preparations or early in vitro harvests in 1/3, and both in 1/3 of cases. GTG-banding was employed in all cases, as were C-banding and RBG- and AgNOR-staining in most cases. Some tumors were near diploid and some near tetraploid, but many had mixed populations of diploid, tetraploid, and octoploid subclones. The most frequent changes were deletions. Loss of 3pl3-pl4, 8p21-p23, or 18q21-q23 was observed in over 60% of tumors, of the inactive Xp in 71% of tumors from females, of the Y in 63% from males, and of 4pl2-pl6, 21q, 9p, and lOp in over 40% of tumors. In our previous studies of SCC of the vulva, 5 of 7 with 18q- died; 2 without 18q- are alive. There was gain (usually 2 - 5x) of lq25-qter, 5p, 7p, and llql3-qter in 35-45% of tumors. Two tumors had an hsr at llql3 which may be associated with amplification of the PRAD1 cyclin gene at that locus. The common breakpoint region in 9p (60% of tumors) appeared to be associated with loss of 9p, an llplS breakpoint in 35% with loss of expression of the UME7 squamous cel 1-specific cell surface antigen, and lcen-lp22 breakpoints in 70% with loss of lp or duplication lq. An unbalanced t (1;acrocentric or 9)(pll;pll) in 30% was usually associated with loss of lp. Complex translocations in 35% were associated with radiotherapy. The emerging karyotypic pattern reflects a constellation of genetic changes in the evolution of SCC and presents a challenge to those who wish to characterize the earliest and the clinically important changes.

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Abstracts - 30th Annual American Cytogenetics Conference


DO CHROMOSOME ABNORMALITIES PREDICT MALIGNANT BEHAVIOR IN MENINGIOMAS? B .F. Schneider, S .R . Vandcnbcrg, K.W . Sudduth, A ND W .L. Golden. Departments o f Pediatrics and Pathology, University o f Virginia, Charlottesville, VA Meningiomas arise within cells covering the brain and spinal cord. Generally these tumors follow a benign clinical course: they are confined within the meninges, thus surgical excision usually results in cure. A small percentage behave aggressively, and recur after gross total resection, or show frank invasion o f brain parenchyma. M eningiomas may be classified into three groups: classical, atypical, and anaplastic. Those with brain invasion are given the additional designation o f malignant. A diagnostic problem with meningiomas is that histopathologic features and biologic behavior may be dissociated. Thus a tumor with malignant brain invasion may display no cytologic atypia. Likewise it is not possible to predict which meningiomas that show focal characteristics o f the atypical category -such as increased cellularity, nuclear pleomorphism, and frequent mitoses - are most likely to have an aggressive clinical course. Characterization o f chromosome abnormalities in the more aggressively behaving meningiomas may help to better predict which will follow a malignant clinical course, and may help to pinpoint genetic changes involved in malignant transformation. This is an ongoing study which to date has yielded chromosome analyses on 10 m eningiomas, 4 o f which are classified as atypical, anaplastic or malignant anaplastic or malignant. Of these latter four, 3 have abnormal karyotypes, as follow s. Case 1: an unbalanced translocation involving chromosomes 3 an 7. Case 2: Two distinct tumor sites were cultured separately. Site 1 had a modal number of 42 with consistent loss o f chromosomes 10, 14, 19 and Y. Site 2 had both structural and numerical abnormalities including the consistent loss of chrom osom es 1 , 7 , 13, 14, 22 and Y, the consistent gain o f chromosomes 5 and 20, and the presence o f three marker chrom osom es. Case 3: a near tetraploid number o f chromosomes with a structurally abnormal chrom osom e 1. Case 3 is classified as malignant because o f brain invasion; however cytologically this tumor appears to be com pletely benign. 31 UTILIZATION OF FLUORESCENCE IN SITU HYBRIDIZATION FOR DELINEATION OF PERICENTRIC CHROM OSOM E BREAKPOINTS. S Schwartz, J Leana-Cox, C Punzalan and B Sullivan. D ivision o f Human G enetics, University o f Maryland School o f M edicine, Baltimore. A small percentage o f non-Robertsonian chromosomal rearrangements involve breakage and reunion in the pericentromeric region which cannot alw ays be precisely identified follow ing conventional banding. We have applied fluorescence in situ hybridization with alphoid centromeric DNA probes to samples from seven patients with structural rearrangements whose breakpoints were not amenable to precise interpretation by conventional banding. These included four from peripheral blood samples [t(I3; 18); t(14; 18), t(l;1 9 ) and t(l;2 2)]; one from an amniotic fluid sample [t(16;22)J; and two from bone marrow samples [t(l;7 ) and t(17;20)]. With one excep­ tion, all the translocations involved either the centromeric regions o f an acrocentric chrom osom e or a chrom osom e with a variable amount o f hctcrochromatic material (e .g ., 1 and 16). In all o f these cases, FISH elucidated the precise breakpoints. N o case appeared to have resulted from fusion o f the tw o centromeres involved in the translocation. H ow ever, in one case the heterochromatic region o f a chrom osom e 1 was split between two chromo­ som es. T w o peripheral blood samples from newborns suspected to have trisomy 18 [t(13; 18) and t(14; 18)] demonstrated a duplication o f 18q. How­ ever, in the familial case, [t(14;18)], the derivative chrom osom e had a chrom osom e 14 centromere, while in the de novo case, [t(13; 18)], the deriva­ tive chrom osom e had a chromosome 18 centromere. These findings may be important in understanding the derivation o f de novo rearrangements. T hese results indicate that FISH can be utilized to: (1) precisely delineate centrom ere origin in structural rearrangements; (2) more accurately determine the amount o f chromosomal material involved in the rearrangement; and (3) gain a better understanding o f the mechanisms leading to rearrangements involving the pericentromeric regions.


DEFINING TH E BREAKPOINT IN RENAL C E L L CARCINOM A USING CH RO M O SO M E M ICRODISSECTION. Leslie Boghosian-Sell, Linda A. Cannizzaro, Dept of Microbiology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA. Consistent chromosome abnormalities of chromosome 3 have been reported in a diversity of tumor types including both hereditary and sporadic forms of renal cell carcinoma (RCC). The breakpoints on chromosome 3 cluster in region 3p 14-21 where loss of heterozygosity for specific alleles has been shown. Cytogenetic and molecular evidence is consistent with the presence of a gene(s) in this region important to neoplasia. We have employed microdissection to more precisely define this region in a hereditary RCC canying a constitutional t (3;8)(p 14.2;q24.1). DNA dissected from both the der(3) and der(8) breakpoint regions was amplified using Alu PCR to generate products in the range of 500 to 1500 bp. Following digestion to remove primers the DNA was clones into pGem 3z. Clones were screened to identify those free of repetitive sequences. Of the initial 5 clones screened, all contained unique sequence. These clones are currently being sequenced and mapped back to the breakpoint region. They will subsequently be used as probes for pulse field analysis to determine their exact location relative to the breakpoint. We hope to generate a large number of probes in this fashion that will enable us to define the breakpoint in RCC and the gene(s) responsible for its etiology.

32 UTILIZATION OF CHROMOSOME-SPECIFIC DNA LIBRARIES TO DELINEATE THE ORIGIN OF DE NOVO DUPLICATIONS. 'J LeanaCox, 7S Levin, !R Surana, 5J Zackowski, and 'S Schwartz. ‘Division o f Human Genetics, University o f Maryland School o f Medicine, Baltimore; ^Walter Reed Army Medical Center, Washington DC; ’University o f Florida, Gainesville. Fluorescence in situ hybridization (FISH) using chromosome-specific DNA libraries provides an effective means o f determining the origin o f extra chromosomal material. This technique was applied to nine cases with de novo chromosomal duplications: 46,X X /46,X X ,I0p + ; 46,X Y ,18q + ; 4 6 ,X X /4 6 ,X X ,2 2 q + ; 46,X X q + ; 4 6 ,X Y ,1 7 q + ; 4 6 ,X Y ,l l q + ; 46,X Y /4 6 ,X Y ,1 2 p + ; 46,X Y ,13p + ; and 47,X Y , + mar. In seven cases, the clinical phenotype or the G-barding pattern o f the duplicated material suggested its most likely chromosomal origin, which was confirmed by FISH with the appropriate chromosome library. In the remaining two cases, careful examination o f the G-banding pattern provided a few alternative sources o f the duplicated material; in each case, the second library utilized yielded conclusive results. Two of the eight duplications (excluding the case with a marker chromosome) were identified as intrachromosomal rearrangements (most likely tandem duplications), while the remaining six consisted o f interchromosomal rearrangements. In two o f these cases, the hybridization patterns delineated the extent o f the duplicated material, which could not be unequivocally determined by G-banding alone. These findings indicate that FISH with chromosome-specific libraries is: (1) effective in confirming the origin o f de novo extra material in patients with diagnostic clinical phenotypes; (2) a valuable addition to routine cytogenetic methods for identifying the source o f de novo duplicated material in patients with nonspecific clinical phenotypes; (3) useful for defining the amount o f de novo duplicated material; and (4) able to provide rapid, unequivocal results in a clinical setting, allowing for appropriate counselling o f patients with these cytogenetic abnormalities. (We acknowledge Imagenetics™ for the gift o f the DNA libraries representing chromosomes 3 ,8 and 12.)

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Abstracts - 30th Annual American Cytogenetics Conference

PARTIAL DUPLICATION 9q and DELETION 6q IDENTIFIED BY CHROMOSOME PAINTING. Nancy Spinner1. Adele Schneider2. Elaine Zackai1' Wendy Golden 3 and Joe Lucas4 . Children’s Hospital of Philadelphia1. Albert Einstein Medical Center. Philadelphia. PA.2 Univ of Virginia3 and Lawrence Livermore National Laboratory 4 Complete definition of abnormal chromosomes is often impossible by conventional cytogenetic techniques. Molecular cytogenetic methods such as chromosome painting provide cytogeneticists with a new approach to defining abnormal chomosomes. We have studied two patients with cytogenetic abnormalities which were incompletely defined by conventional techniques. The first was a newborn with frontal bossing, ridged sutures, deep set eyes with small palpebral fissures, bulbous nose, micrognathia and very long fingers with mild camptodactyly. The karyotype was 46.XY.l2pK, with the extra material consisting of a small G-negative region. Parental chromosomes were normal. Candidate regions for the origin of the extra material were selected based on cytogenetic and clinical criteria, and chromosome painting was done with probes from chromosome 4 and chromosome 9 libraries. The extra band was identified as being derived from chromosome 9. with breakpoint at 9q34 based on the banding pattern. Clinical findings in our pxatient were found to correlate well with features of the few reported cases of trisomy for 9q34 to qter. The second patient was a 4 month old infant with heart anomalies (hypoplastic aortic arch and a VSD), duodenal atresia, malrotation of the gut and dysmorphism Cytogenetic analysis demonstrated 46.XY.6q- hut it was not possible to differentiate between a deletion and the unbalanced product of a translocation. Parental chromosomes were normal. Chromosome painting with probes from a chromosome 6 library showed that the entire shortened chromosome was derived from chromosome 6. Karyotype is 46,XY, del(6)(q25.3-qter). Clarification of these abnormalities is important for comparison of patients with others reported in the literature, to provide accurate prognoses for family members of infants with chromosome abnormalities.

35 DUPLICATION OF 3 q 2 l-> q 2 6 IN A PATIENT WITH Ml ACUTE MYELOGENOUS LEUKEMIA (AML). A. Scalise, C. T heodossiou. N. W isch, V. Najfcld. Tum or Cytogenetic Laboratory, Mount Sinai Medical Center, New York, NY 10029 Chromosomal rearrangements affecting chromosome 3, hand region q21 to q26, such as inversions, translocations or insertions, are associated with those patients (pts.) with AML and myelodysplastic syndiome who show megakaryocyte and platelet abnormalities. We are reporting a pt. with undifferentiated AML whose chromosome analysis of the bone marrow cells showed a terminal deletion 3q21 o f one chromosome 3 and a duplication ol 3q21 to 3q26.2 which was translocated onto the other homolog. A 78-year old female with a 3 year history o f arthritis treated with gold therapy presented with anemia and leukopenia in 1/1987. Bone marrow biopsy cells were negative toi Eacloi VIII. The pt. was admitted on 10/1987 with WCC o f 100 x lO’/L and a platelet count o f 178 x lO’/L. Differential count revealed 80% blasts which weie negative for all tested myeloid and lymphoid reagents with an exceptions ol combined esterase (83% + ) and CDI1 Mo.Ah. (73% + ) A diagnosis o f Ml AML was made and the pt. was started on Ara-C and daunorubicin therapy but expired a month later. Twenty-six Giemsa banded metaphases from the bonemarrow had 4 7 .X X .+ I4,del(3)(q2l),dup(3)(q2l->26.2)t(3:3)(q2l;q26 2) karyotype. The clinical implication o f + 14 in the context o f dup(3q) is unclear One pt. without leukemia but with amegakaryocytic thrombocytopenia showing dup(3)(q24->q26) was recently described (Hallet et al. Brit J Haematol 71:291, 1989). Out o f 3.162 documented pts with AML. one pt had dup(3)(q 1 2 - > q26) and another one had dup(3)(q26->q27) (Milelman 19911. Therefore, our pt. is the third document case with AML and dup(3q) but the duplicated segment 3 q 2 l-> q 2 6 .2 has not been previously described It appeals that band region 3q26 may he crucial in the pathogenesis and therefore dup(.'q) should be considered as a new. although rare, nonrandom rearrangement in AML.

34 ANALYSIS OF LEUKEMIA-ASSOCIATED CHROMOSOMAL REARRANGEMENTS WITH CHROMOSOME-SPECIFIC * DNA LIBRARIES AND FLUORESCENCE IN SITU HYBRIDIZATION, 'BA Sullivan, 'J Leana-Cox, ’C Schiffer, and 'S Schwartz, 'Division of Human Genetics, Departments of OB/GYN and Pediatrics; d ivision of Hematology, Department of Medicine; University o f Maryland School of Medicine, Baltimore. Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries can help to characterize structural chromosome changes by augmenting G-banded interpretations of these aberrations. It has been well established that non-random chromosomal rearrangements are associated with specific leukemias, such as the t(9;22) in chronic myelogenous leukemia, the l(8;21) in acute myelogenous leukemia, and the t(15; 17) in acute promyelocytic leukemia. We have applied FISH with chromosome-specific libraries to analyze both unbanded and previously banded metaphase spreads, ranging in age from one week to several years, of either bone marrow or leukocyte preparations from leukemic patients. Several common leukemia-associated translocations were studied, including; t(9;22), t(8;21), t( 15; 17); as well as their variants: t(3;9;22), t(8;9;22), and t(7;15;17). We intended to determine if this approach was sufficiently sensitive to detect even very subtle rearrangements as well as to clarify the translocations within poorly spread leukemic metaphases. Library and competitor DNA concentrations, as well as hybridization and washing conditions, were optimized for each library. In all cases, the chromosome library studies with FISH confirmed our preli­ minary GTG-banding interpretations, demonstrating that the technique can unequivocally define the chromosomes involved in structural rearrangements. Our results indicate that FISH using chromosome-specific DNA libraries is useful for: 1) analyzing structural aberrations in both previously banded and unhanded preparations o f varying ages; 2) precise diagnosis o f site-specific translocations in leukemia; 3) determining the components o f the transloca­ tions in the presence of poor chromosome morphology and inadequate meta­ phase spreading; 4) defining complex karyotypes; 5) visualizing very subtle chromosomal rearrangements; and 6) confirming G-banded karyotypes. (We acknowledge Imagenetics” for the gift of the chromosomes 3 and 8 probes.)

36 LOSS OF THE Y-CHROMOSOME FROM BONE MARROW CELLS: IS IT AN AGING PHENOMENON OR A MALIGNANCY MARKER? Sofia Shekhter-Levin, Maureen E. Sherer, Niel Wald, and Susanne M. Gollin. Department of Human Genetics, University of Pittsburgh and the Pittsburgh Cancer Institute, Pittsburgh, PA The presence of a 45,X,-Y cell line in the bone marrow of elderly males with normal hemopoiesis was recognized as a normal aging phenomenon. At the same time, data have accumulated suggesting an association between loss of the Y-chromosome with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), chronic myeloproliferative disorders (MPD), chronic lymphoproliferative disorders (LPD), and lymphoma. We studied retrospectively 46 patients who underwent bone marrow aspiration and expressed a clonal loss of the Y-chromosome in bone marrow cells. The distribution of clinical diagnoses was: 11 hospitalized control patients with suspected hematologic malignancies who were later found to have no underlying neoplasia, 9 ANLL patients, 5 CML patients, 11 MDS patients, 8 MPD patients, and 2 patients with LPD. Except for 5 CML patients with a mean age (±SD ) of 55.2 ± 12.8 years, the mean age of the other 30 patients with hematologic disorders was 75.3 ± 8.7 years. This number coincides remarkably with the mean age, 75.4 ± 7.3 years of the 11 control patients. No significant differences in the ratio of mosaic to non-mosaic karyotypes were observed among patients compared to controls. However, the presence of other chromosome changes in addition to Y-chromosome loss was observed in 4 /9 ANLL, 3/5 CML, 2/11 MDS, 3 /8 MPD, and 0/2 LPD patients, but not in controls. Although our numbers are small, on the basis of these data compared to those in the literature, we conclude that loss of the Ychromosome as the sole abnormality in at least MDS and MPD is an age-related phenomenon rather than a malignancy marker.

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30th Annual American Cytogenetics Conference. Virginia Beach, Virginia, March 15-18, 1992. Abstracts.

Cytogenet Cell G enet 6 0 :257-269 (1992) Abstracts of presentations at the Thirtieth Annual American Cytogenetics Conference held on March 15-18, 1...
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