Abstracts of Lectures Gfh ÖGH SGMG Tagungsband Abstracts
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patients were significantly different across pop mosomal level, but not generally at both levels. Abstracts of Lectures ulations with frequency maximum of the com In this view, the aneuploid karyotype is the read mon mutations in EastEurope (W151X, V326L), out of an underlying chromosomal instability NorthWestEurope (IVS81G>C), and SouthEu (CIN). In a small proportion of cancers display 1. Symposia rope (T93M). ing CIN the loss of this checkpoint is associated Carrier frequency analysis of the IVS81G>C, with the mutational inactivation of a human ho W151X, T93M, and V326L mutations in 2250 mologue of the yeast BUB1 gene. BUB1 controls S1 healthy individuals from different European pop mitotic checkpoints and chromosome segrega ulations revealed much higher frequencies for tion in yeast. The Human SHOX Mutation Database these common mutations (e.g. 1:50 for the IVS8 Because the MIN and CIN forms of instability are Beate Niesler, Christine Fischer and Gudrun A. 1G>C in Austria, and 1:84 for the W151X in rarely found to coexist in tumours, it would seem Rappold Poland) than expected from the reported preva that one form of instability is sufficient to drive Institute of Human Genetics, University of lence of the SLOS. Based on these frequencies tumorigenesis. Heidelberg, Im Neuenheimer Feld 328, the expected incidence of SLOS patients with Genetic instability appears early in tumorigene 69120 Heidelberg, Germany null mutations ranges from 1:2000 to 1:16.000. sis and is believed to play a critical role in the The human SHOX gene (Short Stature Home This discrepancy might be due to underdiagno malignant process. Cells with CIN are found to obox gene on the XChromosome) has been iso sis of both severe and mild cases, but also to activate or increase the number of copies of lated by positional cloning and resides in the embryonic/fetal loss of homozygotes for fre oncogenes and lose tumorsuppressor genes, pseudoautosomal region 1 of the sex chromo quent null alleles (W151X, IVS81G>C). whereas cells with MIN accomplish the same somes (Rao et al. 1997, Elison et al. 1997). Het Using eight cSNPs in the DHCR7 gene haplo through mutations in repetitive DNA sequences. erozygousous SHOX mutations have been types were constructed for 52 SLOS chromo This relationship is well documented, and the ef shown to cause idiopathic short stature (ISS), somes. All chromosomes carrying the most fects of genetic instability in cancer develop short stature phenotype in Turner Syndrome (TS) common European mutation IVS81G>C shared ment are straightforward in terms of Darwinian (Rao et al. 1997) and LèriWeill Dyschondrosteo the same haplotype suggesting a founder effect. evolution: Genetic instability provides a reper sis (LWD) (Belin et al. 1998, Shears et al. 1998). Mutations T93M and R404C which involve CpG toire of mutants from which the environment se The homozygous loss of SHOX has been corre islands were found on 4 and 3 different haplo lects favorable variants. Abstracts GfH ÖGH SGMG Tagungsband lated with the Langer type of mesomelic dyspla types respectively suggesting that they are re Recent experiments have shown that CIN and sia (Belin et al. 1998). current. The W151X mutation was present on MIN instabilities reflect resistance to different The Human SHOX Database has recently been different related haplotypes and is probably old carcinogens and that genomic instability in can established to provide clinicians and scientists er than the SNPs used for haplotype construc cers may mirror the mutagenic environments in access to a central source of information about tion indicating that it may be the oldest common which they evolve. It is therefore interesting to all known SHOX mutations associated with short SLOS causing mutation in Europeans. The data speculate that both MIN and CIN develop stature phenotypes. So far, the database con suggest an intriguing high frequency and hetero through selection pressures that can readily be tains 29 unique intragenic mutations of the geneity of the ages and origins of common understood in Darwinian terms rather than aris SHOX gene. These mutations were detected in DHCR7 mutations in ing in a random and mysterious manner. a total of 39 patients from different families. Fourteen of these mutations have been found Functional dissection of signal transduction from the SHOX research group at the Institute of pathways in mammalian cells using RNA Human Genetics in Heidelberg, Germany; 25 S2 interference mutations are from data reported in the litera Anja Schramme, Christine Sers, Oleg ture. Not included in this database are complete Tchernitsa, Katrin Barth and Reinhold Schäfer Genetic instability and darwinian selection SHOX gene deletions which represent the ma Laboratory of Molecular Tumour Pathology, in tumours jority of all detectable SHOX mutations ( Rap Charité, Schumannstr. 20/21, D10117 Berlin Alberto Bardelli pold et al., 2002). The RASRAFMEKERK pathway is the major The Johns Hopkins University, The The database is accessible via the web site player in the regulation of oncogenic signalling Oncology Center, VogelsteinKinzler http://www.shox.unihd.de. It contains general in various types of cells. Most cells express dif Laboratory, Room 520, 1650 Orleans, information about the SHOX gene, allows remote ferent isoforms of the effector kinases down Baltimore MD 21231, USA users to search the data and to submit new mu stream of RAS, however, the contribution of in Whether and how human tumours are genetical tations into the database. The gene structure is dividual kinases is poorly understood. To eluci ly unstable has been debated for decades. There incorporated in order to facilitate data submis date the function of kinase isoforms in control is now evidence that most cancers may indeed sion. ling cell growth and transformation, we have in be genetically unstable, and that the instability References troduced siRNAs (small interfering RNA douplex exists at two distinct levels. 1. Belin et al., 1998. Nat Genet 19:679. es) specific for RAS, RAF, MEK and ERKiso In a small subset of tumours, the instability is 2. Ellison et al. 1997. Hum Mol Genet 8:13417. forms into HRAStransformed rat (FE8) and observed at the nucleotide level and results in 3. Rao E, et al.1997. Nat Genet 16:5463. mouse (NIHpEJ) fibroblasts and into human EJ base substitutions, deletions or insertions of a 4. Rappold etal. JCEM 87: 13841388. bladder carcinoma cells harbouring endogenous few nucleotides. This instability is most easily 5. Shears et al. 1998. Nat Genet 19:703. activated HRAS. HRASspecific siRNA strongly observed at short sequences of DNA repeats inhibited growth in all cells and morphological scattered throughout the genome, called mi transformation in FE8 and NIHpEJ cells. crosatellites, thus generating the characteristic KRASspecific siRNA had no effect. Targeting ki FREQUENCIES AND ORIGINS OF COMMON microsatellite instability (MIN) seen in these tu nases downstream of RAS in mouse NIHpEJ by DHCR7 MUTATIONS CAUSING THE SMITH mours. MIN tumours have nucleotide mutation RNA interference revealed a differential contri LEMLIOPITZ SYNDROME rates two to three orders of magnitude higher bution of MEK1 and MEK2 to growth control. Martina WitschBaumgartner, J. Löffler, M. than normal cells or mismatchrepairproficient Whereas the MEK1specific siRNA inhibited cell Gruber, I. Braun G. Utermann cancers of the same cell type. This form of insta growth only weakly (20%), interference with Institute of Medical Biology and Human bility arises from inactivation of DNA mismatch MEK2 expression resulted in 50% growth sup Genetics, Innsbruck, Austria repair (MMR) genes such as MSH2 or MLH1. pression. Interference with either MEK isoform SmithLemliOpitz syndrome (SLOS [MIM Strikingly, unlike the vast majority of solid tu had almost no effect on cell morphology. How 270400]) is an autosomal recessive multiple con mours, MIN cancers have a normal complement ever, the combination of both siRNAs fully re genital anomaly/mental retardation snydrome of chromosomes. These cancers retain a diploid verted the transformed phenotype of the cells caused by mutations in the D7sterol reductase karyotype ¯ one pair of each of the chromo and inhibited cell growth completely. This indi (DHCR7, E.C.1.3.1.21) gene. Based on case fre somes. cates a strong synergism between MEK1 and quency surveys the incidence of the SLOS has In most other cancers, the instability is observed MEK2. In a similar way, full morphological rever been estimated to range from approximately at the chromosome level, resulting in losses and sion was achieved upon cotransfer of ERK1 and 1:20.000 to 60.000 in populations of European gains of whole chromosomes or large portions ERK2specific siRNAs. Cell growth however, was origin. thereof. NonMIN tumours have a wide variation inhibited up to 50% by either combining ERK1 We are analysing the frequencies, origins, and in chromosome number their karyotypes are and ERK2 siRNAs or after transfer of individual ages of DHCR7 mutations in Europe (Italy, aneuploid. Such observations have led to the siRNAs. Knockdown of kinase isoform expres Spain, Poland, Austria, Germany, and Great suggestion that cancers develop instability ei sion was confirmed by Western blot analysis. In Britain). Mutational spectra analysed in 81 SLOS ther at the sequence level (MIN) or at the chro medgen 14 (2002) 1 hibition of ERK1 resulted in increased phospho S4 cation at the right time. Genome Biol 2, RE rylation of ERK2 and vice versa. This suggests a VIEWS1024. crosstalk