The oncogenic fusion protein-tyrosine kinase ZNF198/fibroblast growth factor receptor-1 has signaling function comparable with interleukin-6 cytokine receptors

The reciprocal t(8;13) chromosome translocation results in a fusion gene (FUS) in which the N-terminal half of the zinc finger protein ZNF198 is combined with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR1). Expression of FUS is suggested to provide growth-promoting activity to myeloid cells similar to the activity of hematopoietic cytokine receptors. This study determined the specificity of FUS to activate signal transduction pathways. Because no tumor cell line expressing FUS was available, the mode of FUS action was identified in cells transiently and stably transfected with an expression vector for FUS. FUS acted as a constitutively active protein-tyrosine kinase and mediated phosphorylation of STAT1, 3, and 5 but not STAT4 and 6. The same specificity but lower activity was determined for normal FGFR1. STAT activation by FUS, similar to that by interleukin-6-type cytokines, promoted STAT-specific induction of genes. The functionality of FUS, as well as the relative recruitment of STAT isoforms, was determined by the dimerizing function of the zinc finger domain. Replacement of the ZNF198 portion by the Bcr portion as present in the t(8;22) translocation shifted the signaling toward a more prominent STAT5 activation. This study documents that both gene partners forming the fusion oncogene define the activity and the signaling specificity of the protein-tyrosine kinase of FGFR1.     

Mapping of four translocation breakpoints within the Duchenne muscular dystrophy gene

There are over 20 females with Duchenne or Becker muscular dystrophy (DMD or BMD) who have X-autosome translocations that break the X chromosome within band Xp21. Several of these translocations have been mapped with genomic probes to regions throughout the large (approximately 2000 kb) DMD gene. In this report, a cDNA clone from the 5' end of the gene was used to further map the breakpoints in four X-autosome translocations. A t(X;21) translocation in a patient with BMD and a t(X;1) translocation in a patient with DMD were found to break within a large 110-kb intron between exons 7 and 8. Two other DMD translocations, t(X;5) and t(X;11), were found to break between the first and the second exon of the gene within a presumably large intron (greater than 100 kb). These results demonstrate that all four translocations have disrupted the DMD gene and make it possible to clone and sequence the breakpoints. This will in turn determine whether these translocations occur by chance in these large introns or whether there are sequences that predispose to translocations.     

Cytogenetic and molecular characterization of eight new reciprocal translocations in the pig species. Estimation of their incidence in French populations

Eight new cases of reciprocal translocation in the domestic pig are described. All the rearrangements were highlighted using GTG banding techniques. Chromosome painting experiments were also carried out to confirm the proposed hypotheses and to accurately locate the breakpoints. Three translocations, rcp(4;6)(q21;p14), rcp(2;6)(p17;q27) and rcp(5;17)(p12;q13) were found in boars siring small litters (8.3 and 7.4 piglets born alive per litter, on average, for translocations 2/6 and 5/17, respectively). The remaining five, rcp(5;8)(p12;q21), rcp(15;17)(q24;q21), rcp(7;8)(q24;p21), rcp(5;8)(p11;p23) and rcp(3;15)(q27;q13) were identified in young boars controlled before entering reproduction. A decrease in prolificacy of 22% was estimated for the 3/15 translocation after reproduction of the boar carrier. A parental origin by inheritance of the translocation was established for the (5;8)(p11;p23) translocation. The overall incidence of reciprocal translocations in the French pig populations over the 2000/2001 period was estimated (0.34%).     

Cloning and mapping of members of the MYM family.

Tandem repeats of a novel, putative, zinc-binding motif (MYM) have been described within the products of two, highly homologous genes: ZNF198/RAMP/FIM and ZNF261/DXS6673E. ZNF198, mapping to 13q11-q12, was recently shown to fuse to the fibroblast growth factor receptor 1 gene in the t(8;13)(p11;q11-q12) rearrangement associated with a stem cell leukemia/lymphoma syndrome. ZNF261 at Xq13.1 is disrupted by a t(X;13)(q13.1;q32) rearrangement in a mentally retarded patient and is a candidate gene for nonspecific X-linked mental retardation. Here we have cloned another member of this family, designated ZNF258, and mapped it to chromosome band 14q12. In addition, ZNF262/KIAA0425 was identified as a further member of the family and mapped to 1p32-p34. The predicted protein products of ZNF258 and ZNF262 maintain the repeats of the MYM motif. Isolation of these new members will facilitate the functional characterization of the MYM family and motif.     

Critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1

The 8p11 myeloproliferative syndrome is an aggressive disorder caused by FGFR1 fusion proteins resulting from a subset of acquired translocations that target chromosome band 8p11. These chimeric proteins have constitutive FGFR1 tyrosine kinase activity and are believed to deregulate hemopoietic development in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here we have studied the role of STAT proteins in transformation mediated by the most common of these fusions, ZNF198-FGFR1. We found that STATs 1, 3, and 5 were activated constitutively in ZNF198-FGFR1-transformed Ba/F3 cells and that STATs 2, 4, and 6 were also tyrosine-phosphorylated. Induction of dominant negative STAT mutants showed that activation of STAT5, but not STATs 1 or 3, was essential for the anti-apoptotic effect of ZNF198-FGFR1 and that STAT5 activation is essential for the elevated levels of BclXL in transformed cells. STAT5 activation was also shown to be required for continued cell cycle progression of BaF3/ZNF198-FGFR1 cells in conditions of cytokine deprivation and for up-regulation of the DNA repair protein Rad51. These findings suggest a critical role of STAT5 activation in transformation mediated by ZNF198-FGFR1.     

Tightly clustered 11q23 and 22q11 breakpoints permit PCR-based detection of the recurrent constitutional t(11;22)

Palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11 at the constitutional t(11;22) breakpoint are predicted to induce genomic instability, which mediates the translocation. A PCR-based translocation-detection system for the t(11;22) has been developed with PCR primers flanking the PATRRs of both chromosomes, to examine the involvement of the PATRRs in the recurrent rearrangement. Forty unrelated carriers of the t(11;22) balanced translocation, plus two additional, independent cases with the supernumerary-der(22) syndrome, were analyzed to compare their translocation breakpoints. Similar translocation-specific junction fragments were obtained from both derivative chromosomes in all 40 carriers of the t(11;22) balanced translocation and from the der(22) in both of the offspring with unbalanced supernumerary-der(22) syndrome, suggesting that the breakpoints in all cases localize within these PATRRs and that the translocation is generated by a similar mechanism. This PCR strategy provides a convenient technique for rapid diagnosis of the translocation, indicating its utility for prenatal and preimplantation diagnosis in families including carriers of the balanced translocation.     

Balanced translocation t(3;18)(p13;q22.3) and points mutation in the ZNF407 gene detected in patients with both moderate non-syndromic intellectual disability and autism

Intellectual disability (ID) is a common disease. While the etiology remains incompletely understood, genetic defects are a major contributor, which include mutations in genes encoding zinc finger proteins. These proteins modulate gene expression via binding to DNA. Consistent with this knowledge, we report here the identification of mutations in the ZNF407 gene in ID/autistic patients. In our study of an ID patient with autism, a reciprocal translocation 46,XY,t(3;18)(p13;q22.3) was detected. By using FISH and long-range PCR approaches, we have precisely mapped the breakpoints associated with this translocation in a gene-free region in chromosome 3 and in the third intron of the ZNF407 gene in chromosome18. The latter reduces ZNF407 expression. Consistent with this observation, in our subsequent investigation of 105 ID/autism patients with similar clinical presentations, two missense mutations Y460C and P1195A were identified. These mutations cause non-conservative amino acid substitutions in the linker regions between individual finger structures. In line with the linker regions being critical for the integrity of zinc finger motifs, both mutations may result in loss of ZNF407 function. Taken together, we demonstrate that mutations in the ZNF407 gene contribute to the pathogenesis of a group of ID patients with autism.     

The constitutional t(17;22): another translocation mediated by palindromic AT-rich repeats

We have demonstrated that the breakpoints of the constitutional t(11;22) are located at palindromic AT-rich repeats (PATRRs) on 11q23 and 22q11. As a mechanism for this recurrent translocation, we proposed that the PATRR forms a cruciform structure that induces the genomic instability leading to the rearrangement. A patient with neurofibromatosis type 1 (NF1) had previously been found to have a constitutional t(17;22) disrupting the NF1 gene on 17q11. We have localized the breakpoint on 22q11 within the 22q11-specific low-copy repeat where the breakpoints of the constitutional t(11;22)s reside, implying a similar palindrome-mediated mechanism for generation of the t(17;22). The NF1 gene contains a 195-bp PATRR within intron 31. We have isolated the junction fragments from both the der(17) and the der(22). The breakpoint on 17q11 is close to the center of the PATRR. A published breakpoint of an additional NF1-afflicted patient with a constitutional t(17;22) is also located close to the center of the same PATRR. Our data lend additional support to the hypothesis that PATRR-mediated genomic instability can lead to a variety of translocations.     

The second case of a t(17;22) in a family with neurofibromatosis type 1: sequence analysis of the breakpoint regions

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22-specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13. Received: 27 August 1996 / Revised: 7 October 1996     

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.     

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.     

Clustered 11q23 and 22q11 breakpoints and 3:1 meiotic malsegregation in multiple unrelated t(11;22) families

The t(11;22) is the only known recurrent, non-Robertsonian constitutional translocation. We have analyzed t(11;22) balanced-translocation carriers from multiple unrelated families by FISH, to localize the t(11;22) breakpoints on both chromosome 11 and chromosome 22. In 23 unrelated balanced-translocation carriers, the breakpoint was localized within a 400-kb interval between D22S788 (N41) and ZNF74, on 22q11. Also, 13 of these 23 carriers were tested with probes from chromosome 11, and, in each, the breakpoint was localized between D11S1340 and APOA1, on 11q23, to a region 相似文献   

A second nonrandom translocation, der(16)t(1;16)(q21;q13), in Ewing sarcoma and peripheral neuroectodermal tumor

The majority of Ewing sarcomas and peripheral neuroectodermal tumors (PNET) that have been karyotyped contain a specific translocation, t(11;22)(q23;q11). We report here a second nonrandom translocation, der(16)t(1;16)(q21;q13), in 2 of 20 cases of Ewing sarcoma (seven previously unreported) and 2 of 7 cases of PNET (all previously unreported). All cases with this translocation also contained the t(11;22). Comparison of C-banding patterns in tumor and peripheral lymphocyte karyotypes in one case indicated that the likely breakpoints were 1q21 and 16q13. The presence of this translocation in cell lines will enable further investigation of the molecular events important in the pathogenesis of Ewing sarcoma and PNET.     

Molecular cloning of Xp11 breakpoints in two unrelated mentally retarded females with X;autosome translocations

Mental retardation is a very common and extremely heterogeneous disorder that affects about 3% of the human population. Its molecular basis is largely unknown, but many loci have been mapped to the X chromosome. We report on two mentally retarded females with X;autosome translocations and breakpoints in Xp11, viz., t(X;17)(p11;p13) and t(X;20)(p11;q13). (Fiber-) FISH analysis assigned the breakpoints to different subbands, Xp11.4 and Xp11.23, separated by approximately 8 Mb. High-resolution mapping of the X- chromosome breakpoints using Southern blot hybridization resulted in the isolation of breakpoint-spanning genomic subclones of 3 kb and 0. 5 kb. The Xp11.4 breakpoint is contained within a single copy sequence, whereas the Xp11.23 breakpoint sequence resembles an L1 repetitive element. Several expressed sequences map close to the breakpoints, but none was found to be inactivated. Therefore, mechanisms other than disruption of X-chromosome genes likely cause the phenotypes.     

A cluster of chromosome 11p13 translocations found via distinct D-D and D-D-J rearrangements of the human T cell receptor delta chain gene.

Human T cell tumours have few consistently occurring translocations which provide markers for this disease. The translocation t(11;14)(p13;q11), however, seems to be an exception, since it has been repeatedly observed in T-ALL. We have analysed a number of T-ALL samples carrying the t(11;14) with a view to assessing the nature of the translocated sequences on chromosomes 11 and 14. Three of the tumours studied have breakpoints, at 14q11, within the T cell receptor delta chain locus, while a fourth appears to break in the J alpha region. The TCR delta sequences involved in the translocation junctions are made from D delta-D delta-J delta joins or from D delta-D delta joins, allowing us to define distinct human D delta and J delta segments. These results allow us to make a comparison between the human and mouse TCR delta loci, both as regards sequence and rearrangement hierarchies. The disparate translocation breakpoints at chromosome 14q11 contrast with the marked clustering of breaks at chromosome 11p13; in all four cases, the breakpoint occurs within a region of less than 0.8 kb of chromosome 11. The analysis of junctional sequences at the 11p13 breakpoint cluster region only shows a consensus heptamer-like sequence in one out of four tumours analysed. Therefore, recombinase-mediated sequence specific recognition is not the only cause of chromosomal translocation.