Ewing's tumour: novel recurrent chromosomal abnormalities demonstrated by molecular cytogenetic analysis of seven cell lines and one primary culture.

Conventional cytogenetics has led to the identification of the primary t(11;22)(q24;q12) translocation in the Ewing's family of tumours, and to the demonstration of certain recurring secondary aberrations that may contribute to neoplastic progression. Other important cytogenetic abnormalities may previously have been overlooked due to the limited resolution of chromosome banding. Here, we have applied the molecular cytogenetic techniques of spectral karyotyping, multiplex-fluorescence in situ hybridisation and comparative genomic hybridisation to the characterisation of seven Ewing's tumour cell lines and one primary culture. These complementary techniques have enabled us to produce a detailed description of the karyotypes of the cell lines and to demonstrate recurring numerical and structural abnormalities. In particular, we have identified a novel, unbalanced translocation involving chromosomes 16 and 17 in three of eight samples, including the primary culture. The unbalanced translocation was associated with comparative genomic hybridisation evidence of loss of 16q and 17p, copy number imbalances that were seen in five and four of the eight samples respectively. Recurrent breakpoints at 16p11.2, 16q11.1, 17p11.2 and 17q11.2 were identified. Our findings indicate that chromosomes 16 and 17 should be investigated further in the search for genes involved in the development of Ewing's family tumours.

Subtyping for TNFa microsatellite sequence variation.

The microsatellite locus TNFa is frequently used as an additional genetic marker in studies of the major histocompatibility complex (MHC). Novel sequence variations at the TNFa locus have been described, and which may have implications for genetic analyses. In this study, we set up a nested polymerase chain reaction-sequence-specific primer (PCR-SSP) approach to type for these TNFa sequence variations. First, sequencing analysis of workshop B lymphoblastoid cell lines (n=13) showed the presence of three sequence variations upstream of the dinucleotide repeat at TNFa. Using nested PCR-SSP, we were able to detect these variations in a larger B lymphoblastoid cell line panel (n=34). Furthermore, we were able to show that TNFa alleles a7 and a10 are present in two distinct conformations leading to "splitting" of TNFa alleles exhibiting identical fragment lengths. To establish the frequency of the TNFa alleles and their variants, we performed microsatellite typing of a large panel of random individuals from the Dutch population (n=272). Subsequent nested PCR-SSP typing showed the presence of three previously described sequence variations in the Dutch population. Furthermore, the presence of a fourth subtype was established. The described variations of allele TNFa7 and TNFa10 are present in the random population with significant frequencies. Haplotyping analysis between HLA-DR, TNFa, and HLA-B showed that allele TNFa7.2 is present in an extended DR7-TNFa7.2-B13 haplotype. In this way, we were able to show that the additional sequence variations behave like distinct TNFa alleles.