Chromosomal breakpoint mapping by arrayCGH using flow-sorted chromosomes.

Despite the recent completion of the human genome project, the mapping of disease-related chromosomal translocation breakpoints and genes has remained laborious. Here, we describe a novel and rapid procedure to map such translocation breakpoints using flow-sorted chromosomes in combination with array-based comparative genomic hybridization (arrayCGH). To test the feasibility of this approach, we used a t(12;15)(q13;q25)-positive cell line with known breakpoint positions as a model. The derivative 12 chromosomes were flow-sorted, labeled, and hybridized to a genome-wide array containing 3648 well-characterized human genomic clones. The exact locations of the breakpoints on both chromosome 12 and 15 could be determined in a single hybridization experiment. In addition, we have tested the minimal amount of material necessary to perform these experiments and show that it is possible to obtain highly reliable profiles using as little as 10,000 flow-sorted chromosomes.

Germ cell tumours in neonates and infants: a distinct subgroup?

Human germ cell tumours (GCTs) constitute a heterogeneous group of tumours that can be classified into four major subgroups. One of these subgroups encompasses (immature) teratomas and yolk sac tumours of patients under the age of 5 years. In this paper we review the various clinical, histological and cytogenetical aspects of these infantile GCTs. The primordial germ cell (PGC) has been suggested to be the cell of origin for GCTs. Infantile GCTs, however, have been suggested to originate from PGCs at a different stage of maturation than adult GCTs. The cytogenetic constitution of infantile GCTs also appears to differ from the adult GCTs and includes recurrent losses of lp and 6q. Recently, two cases of infantile GCT were detected with constitutional 12q13 translocations. These exceptional cases may be instrumental in the search for candidate genes related to infantile and/or adult GCT development.

Comparative proteome, transcriptome, and genome analysis of a gonadal and an extragonadal germ cell tumor cell line.

Whereas clinical differences between testicular and extragonadal germ cell tumors (GCT), like reduced cisplatin sensitivity of extragonadal tumors, are well-established, little is known about underlying tumor biology. A combined approach using global proteome analysis and RT-PCR to assess mRNA levels of selected proteins on the one hand, and array comparative genomic hybridization (array-CGH), on the other hand, was used to compare two germ cell tumor (GCT) cell lines showing embryonal carcinoma histology, one of testicular, and one of extragonadal origin. Overall, the two cell lines show remarkably similar protein profiles. In total, 66 proteins were found to be differentially expressed in an at least 2-fold manner. Of these, 35 proteins (53%) could be positively identified by peptide mass fingerprinting and database search. mRNA levels of 27 differentially expressed proteins were analyzed by RT-PCR. In 17/27 genes (63%), differences in mRNA expression corresponded with differences detected on protein level, suggesting that these proteins are mainly regulated through transcription. Interestingly, no close correlation was found between proteomic and genomic analysis: 13/30 genes (43%) with higher protein levels in one cell line showed higher copy numbers of the respective gene loci in array-CGH analysis. Corresponding differences from proteome, transcriptome, and mRNA analyses were found in 9 of 27 proteins (33%). Several proteins potentially involved in cisplatin resistance were identified in the extragonadal cell line, suggesting that the cisplatin-resistant phenotype of this cell line is multifactorial. Furthermore, our data demonstrate that a combined approach of proteome, transcriptome, and genome analysis is a promising tool to gain information on gene regulation in human tumors.

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines.

Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the U133Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity.

Fusion of the SUMO/Sentrin-specific protease 1 gene SENP1 and the embryonic polarity-related mesoderm development gene MESDC2 in a patient with an infantile teratoma and a constitutional t(12;15)(q13;q25).

Recently, we identified a patient with an infantile sacrococcygeal teratoma and a constitutional t(12;15)(q13;q25). Here, we show that, as a result of this chromosomal translocation, the SUMO/Sentrin-specific protease 1 gene (SENP1) on chromosome 12 and the embryonic polarity-related mesoderm development gene (MESDC2) on chromosome 15 are disrupted and fused. Both reciprocal SENP1-MESDC2 (SEME) and MESDC2-SENP1 (MESE) fusion genes are transcribed in tumor-derived cells and their open reading frames encode aberrant proteins. As a consequence of this, and in contrast to wild-type (WT) MESDC2, the translocation-associated SEME protein is no longer targeted to the endoplasmatic reticulum, leading to a presumed loss-of-function as a chaperone for the WNT co-receptors LRP5 and/or LRP6. Ultimately, this might lead to abnormal development and/or routing of germ cell tumor precursor cells. SUMO, a post-translational modifier, plays an important role in several cellular key processes and is cleaved from its substrates by WT SENP1. Using a PML desumoylation assay, we found that translocation-associated MESE proteins exhibit desumoylation capacities similar to those observed for WT SENP1. We speculate that spatio-temporal disturbances in desumoylating activities during critical stages of embryonic development might have predisposed the patient. Together, the constitutional t(12;15)(q13;q25) translocation revealed two novel candidate genes for neonatal/infantile GCT development: MESDC2 and SENP1.