Distinct MDM2 and P14ARF expression and centrosome amplification in well-differentiated liposarcomas.

Well-differentiated liposarcomas (WDLs) are common soft-tissue tumors in adults. They are characterized by large marker chromosomes and/or ring chromosomes containing 12q-derived sequences in which MDM2 is consistently amplified. WDLs are subdivided into two subtypes according to their karyotype. Type D cells exhibit a near-diploid karyotype, with very few or no chromosome changes. Type H cells exhibit a near-tetraploid karyotype and many structural changes. Expression of P14ARF, MDM2, and TP53 proteins was assayed in the two WDL subtypes to establish whether distinct expression profiles correlated with cell ploidy. Although a transcriptionally functional TP53 was present in most tumors independent of their karyotype, type H cells were characterized by high levels of P14ARF and MDM2 proteins. Although amplified within similar chromosome markers in type D tumors, MDM2 did not appear to be overexpressed. In addition, it was present as a C-terminal truncated protein, indicative of alternatively spliced variants of MDM2 mRNA. As the existence of karyotypically distinct tumors could result from alterations of the mitotic machinery, we investigated the centrosome behavior in the two WDL subtypes. Centrosome amplification occurred in WDL tumors types H and D independent of their ploidy status. Moreover, no functional centrosome difference was found between the two tumor subtypes.

Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants.

Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.

Comparative genomic hybridization reveals novel chromosome deletions in 90 primary soft tissue tumors.

Comparative genomic hybridization (CGH) was used to detect chromosomal gains and losses in a series of 90 frozen soft tissue primary tumors (STTs), all untreated. The material consisted of 69 malignant sarcomas, including 20 malignant fibrous histiocytomas (MFH), 23 liposarcomas (LPS), 6 leiomyosarcomas (LMS), 4 synovial sarcomas, 4 primitive neuroectodermal tumors (PNETs), and various others subtypes, in addition to 21 benign tumors. Within the benign tumors, only 2 of the 3 schwannomas showed genetic changes. In malignant sarcomas, genetic changes were detected in 64 of the 69 samples analyzed (92%), with a mean of 4.5 per sample (range 0-10). Gains and losses on chromosome 13 were observed in 32% of the sarcomas with genomic imbalance. Recurring low-level copy number increases were found at new sites on chromosomes 7 (6 MFH samples, 30%) and 8 (10 LPS samples, 43%), the minimal common regions being 7p15-pter and 8q24. No new recurring high-level amplifications were found. Surprisingly, losses of DNA sequences were more frequent than gains; particularly, losses were the main feature in LMS, with highly recurrent common minimal losses at 11q14-qter and 13q21-q22 (4 samples, 66%, and 5 samples, 83%, respectively). Losses of chromosome 2 sequences (minimal common regions at 2p24-pter and 2q32-qter) were observed in 50% of the MFH analyzed. New recurrent losses of whole or part of chromosome 14 were found in 57% of the pleomorphic LPS (PLPS) analyzed. This study uncovers new clues for the diagnosis of malignant STTs and shows the importance of deletions as events in the early steps involved in the tumorigenesis of STTs.

Structural analysis, expression, and chromosomal localization of the mouse ikba gene.

The pleiotropic transcription factor NF-kappaB is localized in the cytoplasm bound to its inhibitory subunit IkappaB. The predominant form of NF-kappaB is a p50/p65 heterodimer which can be released from IkappaB-alpha and migrate to the nucleus. Previous studies have shown that IkappaB-alpha-/- mice die 8 to 10 days postnatally, showing runting and a severe dermatitis. However, the organ distribution of mouse IkappaB-alpha, the exon-intron structure, and the chromosomal localization of ikba have not been determined so far. A mouse Sv129 genomic DNA library was screened with a human IkappaB-alpha/MAD-3 cDNA probe. One clone (P1) was isolated, spanning the complete ikba gene and the promoter/enhancer region. We show that the exon-intron structure between mouse and pig ikba is completely conserved. In contrast to human ikba, the ankyrin repeat 5 is not interrupted by an intron. Furthermore, the mouse ikba promoter contains 6 putative NF-kappaB binding sequences, which are conserved in mouse, pig, and human, underlining the importance of NF-kappaB as a key regulator of ikba transcription. The deduced amino acid sequence shows >90% similarity between mouse, pig, and human ikba. Chromosome mapping localized the mouse ikba gene to chromosome 12. Northern blot analysis demonstrated predominant expression in lymphoid tissue (lymph node and thymus). However, IkappaB-alpha mRNA was detected as well in liver tissue, the gastrointestinal tract, and the reproductive tract. The cloning and determination of the structure are a prerequisite for the construction of vectors for conditional gene targeting experiments.

Cloning, chromosal mapping, and tissue expression of the gene encoding the human Eph-family kinase ligand ephrin-A2.

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.

Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia.

Translocations in hematologic disease of myeloid or lymphoid origin with breakpoints at chromosome band 12p13 frequently result in rearrangements of the Ets variant gene 6 (ETV6). As a consequence either the ETS DNA-binding domain or the Helix-Loop-Helix (HLH) oligomerization domain of ETV6 is fused to different partner genes. We show here that a t(9;12)(p24;p13) in a case of early pre-B acute lymphoid leukemia and a t(9;15;12)(p24;q15;p13) in atypical chronic myelogenous leukemia in transformation involve the ETV6 gene at 12p13 and the JAK2 gene at 9p24. In each case different fusion mRNAs were found, with only one resulting in an open reading frame for a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the protein tyrosine kinase (PTK) domain of JAK2. The cloning of the complete human JAK2 coding and genomic sequences and of the genomic junction fragments of the translocations allowed a characterization of the different splice events leading to the various mRNAs. JAK2 plays a central role in non-protein tyrosine kinase receptor signaling pathways, which could explain its involvement in malignancies of different hematologic lineages. Besides hop in Drosophila no member of the JAK family has yet been implicated in tumorigenesis.

Framework YAC contig anchored into a 3.2-Mb high-resolution physical map in proximal 11q13.

Despite the presence on band q13 of chromosome 11 of a number of genes predisposing individuals to various human diseases, most of this genomic region remains loosely mapped. Moreover, there is a relative dearth of yeast artificial chromosome (YAC) contigs from genome-wide studies: YACs are irregularly distributed over this chromosomal region and have not been arranged into contigs. We have thus undertaken fine-scale mapping of a 3.2-Mb region flanked by ACTN3 and FGF3. Since this region has demonstrated a high degree of YAC instability, we have established a framework contig by anchoring YACs and cosmids into a high-resolution physical map based on fluorescence in situ hybridization and long-range restriction mapping. The 3.2-Mb area studied includes the boundaries of regions thought to contain genes predisposing individuals to osteoporosis-pseudoglioma syndrome and insulin-dependent diabetes mellitus, as well as genes driving amplification events in human carcinomas. Another feature of this genomic area is that it cross-hybridizes to nonsyntenic regions of the genome. In addition, it spans the region where syntenic conservation with mouse chromosome 19 ends, making clones that we have anchored there valuable tools in understanding genome evolution.

A 5.5-Mb high-resolution integrated map of distal 11q13.

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containing KRN1 and OMP, we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surrounding GARP in breast cancer and for the search of disease genes within this region.

The human inward rectifying K+ channel Kir 2.2 (KCNJ12) gene: gene structure, assignment to chromosome 17p11.1, and identification of a simple tandem repeat polymorphism.

K+ channels are essential for a variety of cellular functions in both excitable and nonexcitable cells, and K+ channel gene alteration has been recently described in cardiac and neurological disorders. To explore further the relations between hereditary human diseases and K+ channels, we isolated from a human cosmid library the gene encoding the inwardly rectifying K+ channel alpha-subunit Kir 2.2 (KCNJ12). PCR analysis performed on this clone indicates that the entire open reading frame is contained in one unique exon. A polymorphic (CA)16 sequence was localized 2.2 kb upstream of the ATG start codon. Fluorescence in situ hybridization on human metaphases assigns the gene to band 17p11.1. The implication of a deletion of the Kir 2.2 gene in the Smith-Magenis syndrome, which is also localized at 17p11, is unlikely since a Kir 2.2-linked microsatellite sequence could be amplified from the DNA of a Smith-Magenis syndrome affected patient bearing a 17p interstitial deletion.

Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma.

Dermatofibrosarcoma protuberans (DP), an infiltrative skin tumour of intermediate malignancy, presents specific features such as reciprocal translocations t(17;22)(q22;q13) and supernumerary ring chromosomes derived from the t(17;22). In this report, the breakpoints from translocations and rings in DP and its juvenile form, giant cell fibroblastoma (GCF), were characterised on the genomic and RNA level. These rearrangements fuse the platelet-derived growth factor B-chain (PDGFB, c-sis proto-oncogene) and the collagen type I alpha 1 (COL1A1) genes. PDGFB has transforming activity and is a potent mitogen for a number of cell types, but its role in oncogenic processes is not fully understood. COL1A1 is a major constituent of the connective tissue matrix. Neither PDGFB nor COL1A1 have so far been implicated in any tumour translocations. These gene fusions delete exon 1 of PDGFB, and release this growth factor from its normal regulation.

Mapping FRA11A, a folate-sensitive fragile site in human chromosome band 11q13.3.

FRA11A, a rare folate-sensitive fragile site assigned to 11q13.3, lies in an area of genomic instability associated with several diseases and amplification events. To map FRA11A, we used fluorescence in situ hybridization with yeast artificial chromosome and cosmid probes on metaphase chromosomes of patients expressing the fragile site. FRA11A was found situated centromeric to ACTN3 and telomeric to D11S913, these markers being within an interval of approximately 1 Mb in the 11q13.3 region.

Detailed map of a region commonly amplified at 11q13-->q14 in human breast carcinoma.

Amplification of loci present on band q13 of human chromosome 11 is a feature of a subset of estrogen receptor positive breast carcinomas prone to metastasis. As many as five distinct amplification units have been described on 11q13. They include particularly a genomic area encompassing the GARP gene at 11q13.5-->q14.1. We have reassessed our current knowledge of this region, located telomeric to CCND1 and EMS1, which is amplified in 7-10% of mammary tumors. The loose definition of the driving forces of these amplification events led us to map accurately the boundaries of the amplifiable region, and thus to contribute a physical and transcriptional map of a 3-Mb region of chromosome 11. Four new genes were placed on the regional map, namely CBP2, CLNS1A, UVRAG, and PAK1. We have narrowed the core of the 11q13-->q14 amplicon to a 350-kb area encompassing D11S533, mostly on its telomeric side. The map reported here represents an indispensable step toward sequencing the entire region, and thus toward uncovering gene(s) which play(s) a critical role in breast cancer progression.

Definition of the Minimal MEN1 Candidate Area Based on a 5-Mb Integrated Map of Proximal 11q13

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with a high penetrance characterized by tumors of the parathyroid glands, the endocrine pancreas, and the anterior pituitary. The MEN1 gene, a putative tumor suppressor gene, has been mapped to a 3- to 8-cM region in chromosome 11q13 but it remains elusive as yet. We have combined the efforts and resources from four laboratories to form the European Consortium on MEN1 with the aims of establishing the genetic and the physical maps of 11q13 and of further narrowing the MEN1 region. A 5-Mb integrated map of the region was established by fluorescence in situ hybridization on both metaphase chromosomes and DNA fibers, by hybridization to DNA from somatic cell hybrids containing various parts of human chromosome 11, by long-range restriction mapping, and by characterization of YACs and cosmids. Polymorphic markers were positioned and ordered by physical mapping and genetic linkage in 86 MEN1 families with 452 affected individuals. Two critical recombinants identified in two affected cases placed the MEN1 gene in an approximately 2-Mb region around PYGM, flanked by D11S1883 and D11S449.

Definition of the minimal MEN1 candidate area based on a 5-Mb integrated map of proximal 11q13. The European Consortium on Men1, (GENEM 1; Groupe d'Etude des Néoplasies Endocriniennes Multiples de type 1).

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with a high penetrance characterized by tumors of the parathyroid glands, the endocrine pancreas, and the anterior pituitary. The MEN1 gene, a putative tumor suppressor gene, has been mapped to a 3- to 8-cM region in chromosome 11q13 but it remains elusive as yet. We have combined the efforts and resources from four laboratories to form the European Consortium on MEN1 with the aims of establishing the genetic and the physical maps of 11q13 and of further narrowing the MEN1 region. A 5-Mb integrated map of the region was established by fluorescence in situ hybridization on both metaphase chromosomes and DNA fibers, by hybridization to DNA from somatic cell hybrids containing various parts of human chromosome 11, by long-range restriction mapping, and by characterization of YACs and cosmids. Polymorphic markers were positioned and ordered by physical mapping and genetic linkage in 86 MEN1 families with 452 affected individuals. Two critical recombinants identified in two affected cases placed the MEN1 gene in an approximately 2-Mb region around PYGM, flanked by D11S1883 and D11S449.

Fluorescence in situ hybridization analysis of t(3; 12)(q26; p13): a recurring chromosomal abnormality involving the TEL gene (ETV6) in myelodysplastic syndromes.

We have identified a new recurrent reciprocal translocation between chromosome 3 and 12 with breakpoints at bands 3q26 and 12p13, t(3;12)(q26;p13) in the malignant cells from five patients with acute transformation of myelodysplastic syndrome or blast crisis of chronic myelogenous leukemia. t(3;12)(q26;p13) appears as a rare but nonrandom event present in various myeloid leukemia subtypes, which is frequently associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and a very poor prognosis. Here, we report the molecular cytogenetic analysis of the t(3;12). Fluorescence in situ hybridization results indicate that the 3q26 breakpoints are quite heterogeneous and occur 5' of MDS1, 3' of EVI1, or between MDS1 and EVI1. Our results are very similar to those observed in other 3q26 rearrangements in which breakpoints were shown to occur over considerable distances 5' and 3' of EVI1. Fluorescence in situ hybridization investigations proved that, in three myelodysplastic syndrome cases with t(3;12)(q26;p13), the 12p 13 breakpoint occurred within the TEL gene.

The 12;21 translocation involving TEL and deletion of the other TEL allele: two frequently associated alterations found in childhood acute lymphoblastic leukemia.

A recurrent t(12;21)(p13;q22) has recently been described in human acute lymphoblastic leukemias (ALLs). This translocation fuses TEL and AML1, two genes previously cloned from translocation breakpoints in myeloid leukemias. In addition, allelic loss of the TEL gene can be detected in 15% to 22% of childhood ALLs. In the present study, we have sought allelic deletions of TEL and the presence of the t(12;21) in 50 children with B-lineage ALL, using a combination of microsatellite typing, fluorescent in situ hybridization (FISH), and analysis of the fusion transcripts resulting from the TEL-AML1 gene fusion. Our results indicate that the association between the t(12;21) and the deletion of the nontranslocated allele of TEL is among the most frequent abnormalities observed in B-lineage ALLs. FISH analysis using several cosmid probes showed that, in one patient with a t(12;21) translocation involving TEL, the second allele had an intragenic deletion. This observation points to TEL as the actual target of 12p12-13 deletions in patients that associate a t(12;21) with a deletion. The TEL-AML1 fusion RNA was found in all patients with the t(12;21) whereas the reciprocal AML1-TEL transcript was only found in a subset of patients, suggesting that only the protein product encoded by TEL-AML1 is likely to play a role in leukemogenesis. The observation that, in two patients with the t(12;21), a deletion of TEL was only present in a subclone indicates that this deletion was a secondary event that occurred after the translocation. The frequent occurrence of TEL deletions in patients with t(12;21) suggests that the deletion of the normal TEL allele subsequent to the t(12;21) provides a further proliferative advantage to leukemic cells.

Rearrangement of proximal 11q13 band in a CMML in acute transformation.

Fluorescence in situ hybridization (FISH) was performed on bone marrow cells thought to contain a t(7;11)(p22;q13) from a patient with chronic myelomonocytic leukemia in transformation. FISH analysis using a panel of 10 probes previously mapped to 11q13 revealed a cytogenetically undetected complex rearrangement that involved chromosomes 7 and 11 as well as a chromosome 3 at band p24. Two distinct translocation breakpoints, both proximal to the BCL1 locus, were found in chromosome 11 that perforce separate it into three subregions. The two breakpoints appear distinct from the two previously described ones which involved the FAU and GSTP1 genes. Our observations add to the involvement of proximal 11q13 in myeloid malignancies.