146 Abstracts

Oral Presentations

"1 A MOUSE MODEL FOR ATAXIA-TELANGIECTASIA: GENETIC MECHANISMS OF TUMORIGENESIS.

C Barlow, M Liyanage, Z Weaver, K Brown, D Tagle, T Ried, and A Wvnshaw-Boris National Center for Human Genome Research, National Institutes of Health, Bethesda, MD 20892

A murine model of ataxia-telangiectasia was created by disrupting the Atm locus via gene targeting. Atm-disrupt~l mice recapitulate the A-T phenotype in humans, providing a mammalian model in which to study the pathophysiology of this pleiotropic disorder. Mice homozygous for the disrupted Atm allele displayed growth retardation, neurologic dysfunction, male and female infertility secondary to the absence of mature gametes, defects in T lymphocyte maturation, and extreme sensitivity to gamma-irradiation. The vast majority of animals developed malignant thymic Iymphomas between two and four months of age. Several consistent chromosomal anomalies were detected in a number of these thymic tumors. The locations of the translocations suggest that the tumors are the result of translocations within the T-cell receptor genes, and may result from the inappropriate expression of oncogenes. Other tumors have also been detected in mutant animals, suggesting that the hiss-of-function of Atm is associated with a general predisposition to tumors. In humans, beterozygous carriers appear to have an increased risk of certain cancers, particularly breast cancer. We are currently investigating whether heterozygous Atm-deficient mice are more susceptible to oncogene-mediated breast tumors and to radiation-induced tumors, hi addition, we are investigating the genetic and biochemical interactions between Atm and proteins that may mediate the effects of Arm on cell cycle checkpoints in response to DNA damage.

2 IDENTIFICATION OF A MAJOR SUSCEPTIIIILITY LOCUS FOR HUMAN PROSTATE CANCER

Trent J M I , S m i t h J R TM , Freije D 2., Carpten JD 1. , Gmnberg H 3., Xu .]2,, ]saacs SD 2, Brownstein MJ 1, Bova GS 2, Gut lq 2, Bujnovszky p2, Nusskem DR 2, Damber JE3, Bergh A 3, Emanuelsson M 3, Kallioniemi OP t , Walker Daniels jl, Bailey-Wilson JE 1 , Beaty TH 2, Meyers DA 2, Walsh PC 2, Collins FS 1 , Isaacs WB 2

INCHGR, NIH, Bethesda, MD 20892 2Johns Hopkins University, School of Medicine, Baltimore, MD 21287 3Umea University, Umea, Sweden

*These authors contributed equally to this study.

Prostate cancer is the most common malignancy diagnosed in U.S. males, accounting for more than 40,000 deaths anually. Despite its high prevalence, and despite reports of familial aggregation of prostate cancer, surprisingly little is known regarding genetic predisposition to this disease. A genome wide scan performed in 66 high-risk prostate cancer families has provided evidence of linkage to the long arm of chromosome 1. Analysis of an additional set of 25 North American and Swedish families using markers in this region resulted in significant evidence of linkage in the complete set of 91 families with a multipoint lod score of 3.67. Allowing for heterogeneity, the multipoint hid score reached 5.43 with ct (the proportion of families linked) estimated as 0.34. These data provide strong evidence of a major prostate cancer susceptibility locus on chromosome 1.

'~ N E W A P P R O A C H E S F O R A N A L Y Z I N G T U M O R C E L L G E N O M E S : M-FISH AND LM-RCA.

MR Speicher, SG Ballard, P Bray-Ward, PM Lizardi and DC Ward. Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510

The ability to identify the twenty-four different human chromosomes in a metaphase spread by the simultaneous hybridization of a set of chromosome- specific DNA probes, each labeled with a different combination of fluorescent dyes, has been reported recently(I,2). These methods, termed multiplex fluorescence in situ hybridization (M-FISH_ and spectral karyotyping (SKY), respectively, have been shown to detect both simple and complex chromosomal abnormalities. The application of M-FISH/SKY in visualizingchromosomal alterations in hematopoietic and solid tumors will be illustrated. A set of YAC clones containing human DNA sequences specific for the subtelomeric region of each chromosome arm also has been assembled and combinatorially labeled such that the telomeric regions of each chromosome are visualized in different colors. The utility of this probe set in detecting cryptic translocations and telomere instability is now being assessed. Potential advantages and disadvantages of M- FISH will be presented and approaches to circumvent the present limitations discussed.

Also under development is a ligase mediated-rolling circle amplification (LM- RCA) method for allele discrimination that can be performed in cytological preparation as well as with DNA in solution or in gridded arrays. An oligonucleotide (70-100mer) is synthesized with sequence homology at each end (18-25 nts) to sequences flanking a 7-12 nucleotide stretch which contains the nucleotide to be interrogated. A gap oligonucleotide is made with either the mutant or wildtype necleotide sequence. These probes are hybridized to target DNA and incubated with a thermostable DNA ligase under conditions that promote ligation of the gap oligonucleotide to both ends of the long otigonucleotide probe only when the gap oligonucleotide is a perfect match to the target DNA. This circularization of the oligonucleotides results in topological constraint to the hybridization site. A primer oligonucleotide, complementary to a portion of the circular oligomer not hybridized to the target sequence, is used with phi29 polymerase, an enzyme with high processivity in second strand synthesis. After traversing the circle once the polymerase strand displaces the primer and newly synthesized second strand via a rolling circle mechanism. Several hundred tandem single-stranded copies of the original circular DNA are made within minutes. Such RCA products can be produced isothermally, in both linear or exponential fashion, and detected by either direct or indirect immunofluorescence. Application of LM- RCA to the detection of point mutations in genes implicated in the pathophysiology of cancer and inherited diseases will be presented.

1MR Speicher, et. aL, Nat. Genet. 12. 368-375 (19960; 2E. Schrtck, et. al, Science 273, 494-497 (1996).

4 SPECTRAL KARYOTYPING Evelin Schrtck Tim Veldman, Hesed Padilla-Nash, Yi Ning, Marek Liyanage, Merryn

Macville, Stan du Manoir, Thomas Ried National Center for Human Genome Research (NCHGR)/NIH, 49 Convent Drive, Bethesda, MD 20892

We have developed a novel approach to identify all human chromosomes labeled in different colors within one fluorescence in situ hybridization experiment. Spectral karyotyping (SKY) complements G-banding in order to identify marker chromosomes, complex chromosomal rearrangements and to elucidate subtle chromosomal aberrations. SKY combines Fourier spectroscopy, CCD-imaging, and optical microscopy to visualize the hybridization of 24 differentially labeled chromosome painting probes (Sehrtck et al., Science). Spectrally overlapping dyes can be resolved unambiguously by measuring emission spectra between 400 and 800 nm simultaneously at all sample points. Chromosome classification based on unique spectra allows to automate karyotype analysis and greatly facilitates the interpretation of complex chromosomal aberrations.

Spectral karyotyping was applied to tumor metaphases obtained from direct preparations, short term cultures and cell lines established from astrocytic tumors, cervix carcinomas, lymphomas and leukemias. In all 30 cases the karyotypes were unambiguously reconstructed, including the identification of marker chromosomes that contained chromosomal aberrations unrecognizable using chromosome banding alone. G-banding and SKY performed subsequently on the same metaphase spreads allow to combine the advantages of both methods. Limitations and further developments of SKY will be discussed.