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Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.

International audience; Recurrent miscarriage (RM) is defined as three or more consecutive pregnancy losses before 24 weeks of gestation. Parental chromosomal abnormalities represent an important etiology of RM. The aim of the present study was to identify the distribution of chromosome abnormalities among Tunisian couples with RM referred to the Department of Cytogenetic at the Pasteur Institute of Tunis (Tunisia) during the last five years. Standard cytogenetic analysis was carried out in a total of 163 couples presenting with two or more spontaneous abortions. Karyotypes were analyzed by R-banding. We identified 14 chromosomal abnormalities including autosomal reciprocal translocation, Robertsonian translocation, inversion, mosaic aneuploidy and heteromorphysm. The overall prevalence of chromosomal abnormalities was 8.5% in our cohort. This finding underlies the importance of cytogenetic investigations in the routine management of RM.

International audience; AIMS: Approximately 20% of soft tissue sarcomas (STS) have subtype-specific chromosomal translocations; these generate chimeric oncoproteins which can act as abnormal transcription factors. Since trabectedin can bind to DNA and displace transcription factors, antitumour activity was explored in translocation-related sarcoma (TRS) subtypes. METHODS: The current retrospective pooled analysis includes data from 81 patients with TRS treated in 8 phase II trials. RESULTS: TRS subtypes were: synovial sarcoma (SS, n=45), myxoid-round cell liposarcoma (MRC-L-sarcoma, n=27), alveolar soft part sarcoma (ASPS, n=4), endometrial stromal sarcoma (ESS, n=3) and clear cell sarcoma (CCS, n=2). All but one patient had received prior chemotherapy (median of 2 lines). Patients received a median of 4 trabectedin cycles (range, 1-48; median dose intensity=0.40 mg/m(2)/week). Partial responses according to Response Evaluation Criteria in Solid Tumours (RECIST) occurred in 8 patients (ORR=10%; 95% CI, 4-19%): four in MRC-L-sarcoma; three in SS and one in ESS. Tumour control rate (ORR plus stable disease) was 59% (95% CI, 48-70%). Median PFS was 4.1 months (6-month PFS rate=40%). Median overall survival was 17.4 months (survival rate at 12 months=60%). Trabectedin had a manageable safety profile. CONCLUSION: Trabectedin demonstrates encouraging disease control in TRS. Since these promising results were generally noted in patients following chemotherapy, a phase III randomised trial in first-line is ongoing to compare trabectedin with doxorubicin-based chemotherapy in patients with TRS.

International audience; Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans and in mice. Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical non-homologous end joining for proper repair, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination, is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes (Rag2(c/c) mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2(c/c) p53(-/-) mice, unlike Rag1(c/c) p53(-/-) and p53(-/-) animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from Atm(-/-) mice. We show that, like ATM-deficiency, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in Rag2(c/c) p53(-/-) and Atm(-/-) mice.

International audience; We sought to understand the genesis of the t(9;22) by characterizing genomic breakpoints in chronic myeloid leukemia (CML) and BCR-ABL-positive acute lymphoblastic leukemia (ALL). BCR-ABL breakpoints were identified in p190 ALL (n=25), p210 ALL (n=25) and p210 CML (n=32); reciprocal breakpoints were identified in 54 cases. No evidence for significant clustering and no association with sequence motifs was found except for a breakpoint deficit in repeat regions within BCR for p210 cases. Comparison of reciprocal breakpoints, however, showed differences in the patterns of deletion/insertions between p190 and p210. To explore the possibility that recombinase-activating gene (RAG) activity might be involved in ALL, we performed extra-chromosomal recombination assays for cases with breakpoints close to potential cryptic recombination signal sequence (cRSS) sites. Of 13 ALL cases tested, 1/10 with p190 and 1/3 with p210 precisely recapitulated the forward BCR-ABL breakpoint and 1/10 with p190 precisely recapitulated the reciprocal breakpoint. In contrast, neither of the p210 CMLs tested showed functional cRSSs. Thus, although the t(9;22) does not arise from aberrant variable (V), joining (J) and diversity (D) (V(D)J) recombination, our data suggest that in a subset of ALL cases RAG might create one of the initiating double-strand breaks.

International audience; Translocations of proto-oncogenes to the B-cell or T-cell antigen receptor loci in acute T- or B-cell leukemia and lymphoma have been, in most cases, accredited to V(D)J or switch recombination depending on the location of the breakpoint at the receptor locus. Only in rare instances, the reports take into account mechanistic characteristics of the translocation mechanism. To assess the functional ability of several sites implicated in supposedly V(D)J-mediated translocations, we tested five sites at four proto-oncogene loci in an ex vivo recombination substrate assay for their potential to act as direct target for V(D)J recombination. Our results show that the LMO2/RBTN2/TTG2 site and one LCK/P56 site readily engage in recombination with a genuine TCR element with the majority of breakpoint junctions showing the characteristics of V(D)J recombination, which strongly supports the involvement of this mechanism in the pathogenesis of the corresponding translocations in vivo. The site at the TLX1/HOX11 locus yielded 0.8% V(D)J-specific junctions. Sites at the LCK/P56 and TCF3/E2A proto-oncogenes resulted in exclusively unspecific breakpoints scattered over part of or the entire proto-oncogene region tested, marking them as unlikely V(D)J recombination targets. Our data suggest that, while being a potentially dangerous mechanism due to the introduction of DNA breaks, V(D)J recombination is a tightly controlled mechanism allowing for only few direct mistakes.

Amphiphilic molecules—molecules that have both hydrophobic and hydrophilic properties—can self-assemble in water to form diverse structures such as micelles, vesicles and tubes1,2,3, and these nanostructures can be used for delivering drugs4,5, stabilizing membrane proteins6 or as nanoreactors7. We have previously shown that lipids can self-organize on the surface of single-walled carbon nanotubes into regular ring-shaped assemblies8. Here we show that these lipid assemblies can be polymerized and isolated from the nanotube template by application of an electric field. We also demonstrate that these assemblies are monodispersed, water-soluble, and can dissolve various hydrophobic rylene dyes, fullerenes and membrane proteins. The stability of these constructs and their diverse applications will be useful in the fields of cosmetics, medicine and material sciences. Carbon nanotubes used as templates for polymerizing lipids into regular ring-shaped water-soluble assemblies that can dissolve various hydrophobic compounds and membrane proteins, could have applications in cosmetics, medicine and materials science.

We have investigated the breakpoints of a balanced reciprocal translocation between chromosomes X and 5, [46,X,t(X;5)(p11.1;q31.1)], in a woman with mild mental retardation (MR). Methylation studies showed a 100% skewed X-inactivation in patient-derived lymphocytes. Cloning and sequencing of the junction fragment from the X derivative showed that the breakpoint occurred in intron 3 of the CDKL3 gene on chromosome 5 and in a region devoid of genes on chromosome X. Quantitative RT-PCR analyses on patient-derived lymphoblastoid cells documented a significant 50% decrease of the CDKL3 transcript level. Allelic expression analysis, using an intronic SNP that was RT-PCR amplified from CDKL3 pre-mRNA, provided further evidence that the CDKL3 gene was transcribed from only one allele. Decreased CDKL3 gene expression was definitively confirmed at the protein level by immunoblot analysis. CDKL3 is a member of a subset of the cdc2-related protein kinase family that shows similarity to both mitogen-activated protein kinases (MAPK) and cyclin-dependant kinases (cdks). Importantly, one member of the family, CDKL5, has been implicated in atypical Rett syndrome, West syndrome, and X-linked infantile spasm, all including MR as a manifestation. Expression studies demonstrated that the mouse homologue, mCdkl3, was expressed in all brain regions investigated and throughout mouse development, a pattern that is consistent with a role in development and brain function. Together the data suggest that haploinsufficiency of CDKL3 in the t(X;5) patient contributes to her phenotype, and that the CDKL3 gene is a strong candidate for nonsyndromal autosomal dominant MR.

International audience; MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.

International audience; The t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation.

International audience; Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

A large number of lymphoid malignancies is characterized by specific chromosomal translocations, which are closely linked to the initial steps of pathogenesis. The hallmark of these translocations is the ectopic activation of a silent proto-oncogene through its relocation at the vicinity of an active regulatory element. Due to the unique feature of lymphoid cells to somatically rearrange and mutate receptor genes, and to the corresponding strong activity of the immune enhancers/promoters at that stage of cell development, B- and T-cell differentiation pathways represent propitious targets for chromosomal translocations and oncogene activation. Recent progress in the understanding of the V(D)J recombination process has allowed a more accurate definition of the translocation mechanisms involved, and has revealed that V(D)J-mediated translocations result both from targeting mistakes of the recombinase, and from illegitimate repair of the V(D)J recombination intermediates. Surprisingly, V(D)J-mediated translocations turn out to be restricted to two specific sub-types of lymphoid malignancies, T-cell acute lymphoblastic leukemias, and a restricted set of mature B-cell Non-Hodgkin's lymphomas.

Follicular lymphoma (FL) is one of the most common B-cell lymphoma, and remains virtually incurable despite its relatively indolent nature. T(14;18)(q32;q21), the genetic hallmark and early initiating event of FL pathogenesis, is also present at low frequency (10−5–10−7) in blood from healthy individuals (HI), indicating that t(14;18) and the ensuing BCL2 overexpression is necessary but not sufficient for malignant transformation. It has long been assumed that in HI, t(14;18) is carried by circulating quiescent naïve B-cells, where its oncogenic potential would be restrained. Yet, several reports, including long-term persistence and immunomodulation of t(14;18)+ cells in lymphoma-free individuals, led us to question this model and investigate the status of circulating t(14;18)+ cells in HI. We first determined if t(14;18)+ cells are naïve B-cells by assessing class-switch recombination (CSR) on the translocated allele. Using 2 long-range PCR assays designed to amplify unswitched BCL2/Sμ and switched BCL2/Sg regions, DNA samples from 6 HI with t(14;18) were tested. Contrary to previous assumptions, our data clearly show that most peripheral t(14;18)+ cells already underwent CSR (n=5/6) and therefore that most t(14;18)+ cells are not naïve B-cells. Are they then memory B cells? Naïve and memory B cell subsets from 9 HI were isolated by cell sorting according to IgD and CD27 markers, and the rate of t(14;18) analyzed in each subset relatively to that of the total B cells. Strikingly, while the level of naïve t(14;18)+ cells remained at baseline for all individuals, memory B-cells tightly accounted for the wide modulation of t(14;18) frequencies observed between individuals. In addition, sequence analysis of t(14;18) clones revealed that this wide modulation was not due to the accumulation of clonally unrelated t(14;18) naïve B-cells, but rather to the clonal expansion of t(14;18)-bearing memory B-cells. To further define the t(14;18)+ cells, we next examined the repartition of the translocation in the IgD−/CD27+ and IgD+/CD27+ memory B-cell subsets. Unexpectedly, we found that the IgD+/CD27+ subset contained significantly higher rates of translocation than the IgD−/CD27+, both in terms of prevalence and frequency. Thus, while CSR is found in the majority of translocated alleles (~75%), most t(14;18)+ memory B cells have not switched their productive allele (~70%) and express an IgM/D. Most importantly, although atypical among physiological peripheral B-cells, this “allelic paradox” is a specific hallmark of FL, and suggests the presence of the same selective pressure in favor of sIgM expression on a B-cell population that is at the same time permanently driven to switch. In line with B-cell hyperplasia in BCL2 transgenic mice slowly progressing to low grade lymphoma, it is likely that “FL-like” cells in HI are rescued by BCL2 from apoptosis, and “frozen” at a differentiation stage in which constitutive AID expression drives continuous somatic hypermutation and CSR activity, two mechanisms conferring a high propensity for genomic instability. Altogether, our findings identify a novel intermediate step in early lymphomagenesis, and strongly impact both on the current understanding of disease progression from potent pre-malignant niches, and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

International audience; Hypomagnesemia with secondary hypocalcemia (HSH) is a rare inherited disease, characterised by neurological symptoms, such as tetany, muscle spasms and seizures, due to hypocalcemia. It has been suggested that HSH is genetically heterogeneous, but only one causative gene, TRPM6, on chromosome 9 has so far been isolated. We have now studied the genetic background of HSH in four Polish patients belonging to three families, and a HSH patient carrying an apparently balanced X;9 translocation. The translocation patient has long been considered as an example of the X-linked form of HSH. We identified six TRPM6 gene mutations, of which five were novel, in the Polish patients. All the alterations were either nonsense/splicing or missense mutations. The clinical picture of the patients was similar to the HSH patients reported earlier. No genotype-phenotype correlation could be detected. Sequencing did not reveal any TRPM6 or TRPM7 gene mutations in the female HSH patient with an X;9 translocation. Isolation of the translocation breakpoint showed that the chromosome 9 specific breakpoint mapped within satellite III repeat sequence. The X-chromosomal breakpoint was localised to the first intron of the vascular endothelial growth factor gene, VEGFD. No other sequence alterations were observed within the VEGFD gene. Even though the VEGFD gene was interrupted by the X;9 translocation, it seems unlikely that VEGFD is causing the translocation patient's HSH-like phenotype. Furthermore, re-evaluation of patient's clinical symptoms suggests that she did not have a typical HSH.

International audience; DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

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International audience; The Tat (twin-arginine translocation) system is a protein targeting pathway utilized by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli, Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. The present study demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterization suggested that the C-terminal 'Domain IV' of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.

International audience; Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.

International audience; BACKGROUND: Genetic abnormalities are common in patients with multiple myeloma, and may deregulate gene products involved in tumor survival, proliferation, metabolism and drug resistance. In particular, translocations may result in a high expression of targeted genes (termed spike expression) in tumor cells. We identified spike genes in multiple myeloma cells of patients with newly-diagnosed myeloma and investigated their prognostic value. DESIGN AND METHODS: Genes with a spike expression in multiple myeloma cells were picked up using box plot probe set signal distribution and two selection filters. RESULTS: In a cohort of 206 newly diagnosed patients with multiple myeloma, 2587 genes/expressed sequence tags with a spike expression were identified. Some spike genes were associated with some transcription factors such as MAF or MMSET and with known recurrent translocations as expected. Spike genes were not associated with increased DNA copy number and for a majority of them, involved unknown mechanisms. Of spiked genes, 36.7% clustered significantly in 149 out of 862 documented chromosome (sub)bands, of which 53 had prognostic value (35 bad, 18 good). Their prognostic value was summarized with a spike band score that delineated 23.8% of patients with a poor median overall survival (27.4 months versus not reached, P

International audience; Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell-specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4-10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome.

Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1., Author Summary Chromosomal rearrangements are associated with many tumor types, as they are one way in which genes affecting cancer initiation and progression become mutated. One type of rearrangement is a chromosomal translocation, in which parts of two different chromosomes join together. Although infrequent, translocations occur when both chromosomes undergo breakage and the ends from different chromosomes join rather than the two ends from the same chromosome. Human and mouse cells have three known DNA ligases which catalyze the joining of DNA ends (Lig1, Lig3, and Lig4). Lig4 is important for joining the correct ends together, thereby suppressing translocations. In this report, the role of the other two DNA ligases is examined in a novel mouse cell system. Lig3 is found to be required for efficient chromosomal translocation formation, but in its absence Lig1 can substitute, although less efficiently and although the joining characteristics of the two DNA ligases differ. These studies define the hierarchy of the three DNA ligases in this type of genomic rearrangement.

International audience; Follicular lymphoma (FL) pathogenesis is a complex and fascinating multi-hit process, escalating along successive derailments of the distinctive molecular and cellular mechanisms paving B-cell differentiation and activation. This progressive subversion of B-cell receptor diversification mechanisms and B-cell homeostasis likely occurs during a protracted preclinical phase of asymptomatic growth, in which premalignant clones already disseminate and establish "niches" in secondary lymphoid organs. Following FL diagnosis, a parallel indolent behavior is observed in most patients, slowly progressing over a period of many years, to eventually generate a highly refractory (and in some case transform into an aggressive subtype of) lymphoma. Novel insights in human germinal center B-cell biology recently allowed a more comprehensive understanding of the various illegitimate events sequentially involved in the premalignant progression phases. In this review, we will discuss how these new data have modified our perception of early FL pathogenesis, the new questions and challenges it opened up, and how this knowledge could impact on innovative programs of early detection, follow-up, and patient management.

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International audience; Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumours--those targeting 1q12 satellite DNA--can directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range 'pairing' between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms.

International audience; Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and rarely occurs in adults. There are six main subtypes, each histologically, clinically, and cytogenetically distinct. Embryonal RMS is characterized by chromosomal gains, usually not associated with any consistent structural anomaly. We describe here a case of embryonal RMS in a 19-year-old female patient. The conventional cytogenetic analysis showed a t(4;22)(q35;q12) translocation as the sole cytogenetic change. Complementary fluorescence in situ hybridization analysis showed that the translocation breakpoints were located in the EWSR1 gene at 22q12 and the region of the DUX4 and FSHMD1A at 4q35. This constitutes a novel example of the high frequency of EWSR1 rearrangements in various types of sarcomas as well as of its ability to fuse with a large variety of partner genes. Because DUX4 is involved in myogenic differentiation and cell-cycle control, the striated muscle differentiation observed in the present case might be a direct consequence of the alteration of the DUX4 region generated by the t(4;22). The involvement of the DUX4 region might represent the genetic hallmark of a novel subclass of small round cell tumors.

International audience; The t(14;18) translocation constitutes the initiating event of a causative cascade leading to follicular lymphoma (FL). t(14;18) translocations are present in blood from healthy individuals, but there is a trend of increased prevalence in farmers exposed to pesticides, a group recently associated with higher risk of t(14;18)(+) non-Hodgkin's lymphoma development. A direct connection between agricultural pesticide use, t(14;18) in blood, and malignant progression, however, has not yet been demonstrated. We followed t(14;18) clonal evolution over 9 yr in a cohort of farmers exposed to pesticides. We show that exposed individuals bear particularly high t(14;18) frequencies in blood because of a dramatic clonal expansion of activated t(14;18)(+) B cells. We further demonstrate that such t(14;18)(+) clones recapitulate the hallmark features of developmentally blocked FL cells, with some displaying aberrant activation-induced cytidine deaminase activity linked to malignant progression. Collectively, our data establish that expanded t(14;18)(+) clones constitute bona fide precursors at various stages of FL development, and provide a molecular connection between agricultural pesticide exposure, t(14;18) frequency in blood, and clonal progression.

t(11;14)-positive clones can persist over a long period of time in the peripheral blood of healthy individuals

International audience; Rare cases of peripheral T-cell lymphomas with follicular growth pattern (PTCL-F) have been recently reported, and their association with t(5;9)(q33;q22) involving ITK and SYK has been suggested. However, the clinicopathologic aspects of PTCL-F are poorly described and the normal cell counterpart of this subgroup of lymphoma is still unknown. Therefore, we analyzed the pathologic, phenotypic, and cytogenetic features of a series of 30 patients (range: 33 to 88 y) that showed histopathologic features of PTCL-F in at least 1 biopsy (n=30), either at initial presentation (n=26) or at relapse (n=4). Neoplastic cells were medium-sized clear cells that were CD4+ (24/27, 89%), CD10+ (21/29, 72%), BCL-6+ (14/19, 74%), and expressed programed death-1 (27/27, 100%), CXCL13 (23/27, 85%), and ICOS (11/11, 100%), markers of follicular helper T cells (TFH). Four of 22 patients (18%) had t(5;9)(q33;q22) detected by fluorescence in situ hybridization. Patients with clinical data available had multiple lymphadenopathies (25/28, 89%), stage III to IV diseases (17/26, 65%), B symptoms (7/27, 26%), and skin lesions (6/23, 26%). Three patients with sequential biopsies disclosed clinical and histopathologic features of angioimmunoblastic T-cell lymphoma at initial presentation. Our results show that this rare form of PTCL-F (1) has an immunophenotype indicative of derivation from TFH cells, (2) is associated with t(5;9) in a proportion of cases, and (3) shows some overlapping features with angioimmunoblastic T-cell lymphoma, raising the question of a possible relationship.

The allotetraploid species Aegilops variabilis Eig (2n = 28, UUSvSv) belongs to the tribe Triticeae and is closely related to wheat. One accession, Ae. variabilis No. 1, was found to be resistant to the cereal cyst nematode (CCN) and the root-knot nematode (RKN). As the genetic variability for resistance to those two pests is limited within wheat, this accession was crossed to bread wheat. Previous work enabled the development of two addition lines and two translocation lines carrying resistance. Here, we demonstrate, using genomic in situ hybridization, that there is no U–Sv interchange in the parental accession of Ae. variabilis. However, there are multiple rearrangements in the Sv chromosomes. The Ae. variabilis chromosome carrying the CreX gene for resistance to CCN combined segments with homoeology to wheat groups 1, 2, 4, and 6. The CreX gene belongs to the group 1 part and it was likely to have been introduced into chromosome 1BL at a similar location as the previously found QTL QCre.srd-1B for CCN resistance. The second Ae. variabilis chromosome carrying CreY and Rkn2 combined segments with homoeology to wheat groups 2, 4, and 7 on its short arm and group 3 on its long arm. It was designated as 3Sv. The two genes for resistance are carried by its long arm and have been transferred to wheat chromosome 3BL through homoeologous and genetically balanced recombination. Different SSR markers present in the introgressed segments could be used in marker-assisted selection.

International audience; Rituximab, an anti-CD20 monoclonal antibody, is widely used in the treatment of B-cell lymphoma. Some reports have outlined histologic modifications in bone marrow specimens from patients treated with this antibody, notably the presence of CD3(+) lymphoid aggregates morphologically mimicking residual lymphoma. To gain insight into the significance of such infiltrates, serial BM trephines obtained in 39 patients with B-cell follicular lymphoma treated by rituximab and enrolled in the GOELAMS-GELA intergroup FL2000 protocol were reexamined. The 39 patients were 22 women and 17 men with a median age of 50 years (range, 29-75 years). All pretreatment bone marrow biopsies showed CD20(+) lymphomatous cells. A second biopsy was obtained between 30 and 100 days after the last rituximab injection: 19 (48%) were morphologically diagnosed as negative (no lymphoid infiltrates or only minor lymphoid aggregates) and 20 (51%) as positive because of persistent lymphoid nodules. After immunohistochemical analysis, 13 (33%) cases were reinterpreted as false-positive because of the complete absence of CD20(+) cells, with the lymphoid nodules consisting of CD3(+) and CD5(+) T cells. Most of them also expressed CD4(+), whereas only a few CD8(+) cells were present. Among these 13 false-positive cases, 12 were BCL2-IGH polymerase chain reaction-negative in the bone marrow aspirate at the time of biopsy. The 13th case turned out to be negative in the 18th-month bone marrow aspirate. In all of these cases, lymphoid aggregates had disappeared on bone marrow biopsies performed 18 months after treatment. After a mean follow-up of 4.5 years, 9 of 13 patients were in remission as compared with only 2 among the 7 patients with postrituximab persistent CD20(+) lymphomatous cells. There was no statistically significant difference between this false-positive group of patients and that with negative postrituximab bone marrow regarding sex, age, medullar involvement pattern before treatment, delay between rituximab treatment, and molecular status. Interestingly, we noted a more favorable outcome (70% versus 52% remission) for the false-positive cases, suggesting that these T-cell reactions could be the hallmark of specific antitumoral immunity after rituximab treatment and should be properly investigated.

It has long been thought that signal joints, the byproducts of V(D)J recombination, are not involved in the dynamics of the rearrangement process. Evidence has now started to accumulate that this is not the case, and that signal joints play unsuspected roles in events that might compromise genomic integrity. Here we show both ex vivo and in vivo that the episomal circles excised during the normal process of receptor gene rearrangement may be reintegrated into the genome through trans-V(D)J recombination occurring between the episomal signal joint and an immunoglobulin/T-cell receptor target. We further demonstrate that cryptic recombination sites involved in T-cell acute lymphoblastic leukemia–associated chromosomal translocations constitute hotspots of insertion. Eventually, the identification of two in vivo cases associating episomal reintegration and chromosomal translocation suggests that reintegration events are linked to genomic instability. Altogether, our data suggest that V(D)J-mediated reintegration of episomal circles, an event likely eluding classical cytogenetic screenings, might represent an additional potent source of genomic instability and lymphoid cancer., Author Summary Lymphoid cells recognize billions of pathogens as a result of gene rearrangements that generate pathogen-specific B- and T-cell receptors. This genetic reshuffling, called V(D)J recombination, occasionally misfires and damages genomic integrity. When such aberrations dysregulate proto-oncogenes, cancer ensues. It has become increasingly clear that multiple oncogenes acting in different cellular pathways can cooperate to cause cancer. Nevertheless, in the case of T-cell acute lymphoblastic leukemia, about a third of cases display oncogene activation in the absence of identified aberration, suggesting the presence of additional mechanisms of chromosomal alteration. In the hunt for such mechanisms, episomal circles (DNA segments that are excised during V(D)J recombination) have recently drawn attention. Moreover, signal joints, short sequences formed after gene rearrangements, once considered harmless, now appear to take part in events that might compromise genomic integrity. Using ex vivo recombination assays and genetically modified mice, we demonstrate that episomal circles may be reintegrated into the genome through recombination occurring between the episomal signal joints and a T-cell receptor target. Furthermore, we show that cryptic recombination sites located in the vicinity of oncogenes constitute hotspots of episomal insertion. Altogether, our results suggest that reintegration of excised episomal circles constitute a potential source of genomic instability and cancer in leukemia and lymphoma., Episomal DNA circles are the by-products of immunoreceptor gene rearrangements in lymphoid cells. Episomal circles can be reintegrated into the genome by trans-V(D)J recombination and cause oncogene deregulation.

Methot et al. identify a mechanism for cytoplasmic retention of activation-induced deaminase (AID) in cells. Interactions of AID with Hsp90 and eEF1A proteins, both of which stabilize AID, promote sequential folding and retention of functional AID in the cytoplasm. Inhibition of the translation elongation factor eEF1A blocks its interaction with AID, which then accumulates in the nucleus, increasing class switch recombination and the generation of chromosomal translocation byproducts., Activation-induced deaminase (AID) initiates mutagenic pathways to diversify the antibody genes during immune responses. The access of AID to the nucleus is limited by CRM1-mediated nuclear export and by an uncharacterized mechanism of cytoplasmic retention. Here, we define a conformational motif in AID that dictates its cytoplasmic retention and demonstrate that the translation elongation factor eukaryotic elongation factor 1 α (eEF1A) is necessary for AID cytoplasmic sequestering. The mechanism is independent of protein synthesis but dependent on a tRNA-free form of eEF1A. Inhibiting eEF1A prevents the interaction with AID, which accumulates in the nucleus and increases class switch recombination as well as chromosomal translocation byproducts. Most AID is associated to unspecified cytoplasmic complexes. We find that the interactions of AID with eEF1A and heat-shock protein 90 kD (HSP90) are inversely correlated. Despite both interactions stabilizing AID, the nature of the AID fractions associated with HSP90 or eEF1A are different, defining two complexes that sequentially produce and store functional AID in the cytoplasm. In addition, nuclear export and cytoplasmic retention cooperate to exclude AID from the nucleus but might not be functionally equivalent. Our results elucidate the molecular basis of AID cytoplasmic retention, define its functional relevance and distinguish it from other mechanisms regulating AID.

International audience; Adult patients with acute lymphoblastic leukemia (ALL) and t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4 have a poor outcome. We have evaluated the impact of an intensified post-remission therapy using a high-dose chemotherapy course followed by allogeneic or autologous SCT on the outcome of 58 patients with t(1;19)/E2A-PBX1 (E2A group, n=24) or t(4;11)/MLL-AF4 (MLL group, n=34) treated in the LALA-94 multicenter prospective study. Patients in the MLL group had higher WBC counts and more frequent DIC. CR rates achieved by MLL and E2A groups were similar to other B-cell ALL (87, 82 and 86% respectively). While in CR, patients with a donor were assigned to alloSCT (n=22), the remaining patients with were randomized between autoSCT (n=15) or chemotherapy (n=8). Five-year overall survival was 31 and 45% for E2A and MLL groups, respectively. In both groups, DFS was higher in the alloSCT arm as compared to autoSCT and chemotherapy arms. The results of this study show that chemotherapy intensification did not overcome the poor prognosis of adults with t(1;19)/E2A-PBX1. Allogeneic SCT should thus be offered in first CR to patients with t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4. New therapeutic approaches are needed for patients without donor.

Made available in DSpace on 2019-09-12T16:53:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2006 Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) The extensive heterogeneity underlying the genetic component of mental retardation (MR) is the main cause for our limited understanding of the aetiology of this highly prevalent condition. Hence we set out to identify genes involved in MR. We investigated the breakpoints of two balanced X;autosome translocations in two unrelated female patients with mild/moderate MR and found that the Xp11.2 breakpoints disrupt the novel human KIAA1202 (hKIAA1202) gene in both cases. We also identified a missense exchange in this gene, segregating with the Stocco dos Santos XLMR syndrome in a large four-generation pedigree but absent in > 1,000 control X-chromosomes. Among other phenotypic characteristics, the affected males in this family present with severe MR, delayed or no speech, seizures and hyperactivity. Molecular studies of hKIAA1202 determined its genomic organisation, its expression throughout the brain and the regulation of expression of its mouse homologue during development. Transient expression of the wild-type KIAA1202 protein in HeLa cells showed partial colocalisation with the F-actin based cytoskeleton. On the basis of its domain structure, we argue that hKIAA1202 is a new member of the APX/Shroom protein family. Members of this family contain a PDZ and two ASD domains of unknown function and have been shown to localise at the cytoskeleton, and play a role in neurulation, cellular architecture, actin remodelling and ion channel function. Our results suggest that hKIAA1202 may be important in cognitive function and/or development. Max Planck Inst Mol Genet, Dept Human Mol Genet, D-14195 Berlin, Germany; ULP, INSERM, CNRS, Inst Genet & Biol Mol & Cellularie,Dept Mol Patho, Illkirch Graffenstaden, France; Greenwood Genet Ctr, JC Self Res Inst, Greenwood, SC 29646 USA; Univ Ulm, Dept Human Genet, Ulm, Germany; Wilhelm Johannsen Ctr Funct Genome Res, Dept Med Biochem & Genet, Copenhagen, Denmark; INSERM, Serv Genet, Tours, France; Katholieke Univ Leuven Hosp, Ctr Human Genet, B-3000 Louvain, Belgium; CNRS, INSERM, Inst Cochin Genet Mol, Paris, France; Univ Nijmegen St Radboud Hosp, Med Ctr, Dept Human Genet, Nijmegen, Netherlands; Univ Adelaide, Dept Med Genet, Womens & Childrens Hosp, Adelaide, SA, Australia; Univ Adelaide, Dept Pediat, Adelaide, SA, Australia; Hop Univ Strasbourg, Serv Genet Med, Strasbourg, France; Universidade de Taubaté (Unitau), Inst Butantan, Genet Lab, Sao Paulo, Brazil; Erasmus MC, Dept Genet, Rotterdam, Netherlands

Objective To investigate the existence of an interchromosomal effect in balanced reciprocal translocation carriers and to evaluate their risk of having an affected child or repeated spontaneous abortions. Design Analysis of chromosomal sperm content by fluorescence in situ hybridization (FISH) using probes for chromosomes 1, 15, 16, 17, 18, X, and Y. Setting University hospital. Patient(s) Six male carriers of balanced chromosome rearrangements, one with normal sperm parameters and five with oligoasthenoteratozoospermia, and seven fertile controls. Intervention(s) Fluorescence in situ hybridization for chromosomal enumeration on sperm samples. Main Outcome Measure(s) Rate of disomy for the studied chromosomes. Result(s) A total of 123,842 spermatozoa were scored (82,181 for controls and 41,661 for patients). For each patient, at least one chromosome studied presented a significantly increased rate of disomy. One patient showed a clear interchromosomal effect for at least three of the six chromosomes studied. Disomy rates were statistically significantly and inversely correlated with the total progressive motility of spermatozoa. Conclusion(s) The observed interchromosomal effect seems to be translocation, patient, and chromosome dependent. Variable effects were observed, according to the chromosome studied. When we looked at patients carrying the same translocation, the level of disomy rate was variable and affected different chromosomes.

The frequent rearrangement of chromosome band 1q12 constitutive heterochromatin in hematologic malignancies suggests that this rearrangement plays an important pathogenetic role in these diseases. The oncogenic mechanisms linked to 1q12 heterochromatin are unknown. Constitutive heterochromatin can epigenetically regulate gene function through the formation of transcriptional-silencing compartments. Thus, as a first step toward understanding whether 1q12 rearrangements might compromise such activity in tumor cells, we investigated the 3-D organization of the 1q12 heterochromatin domain (1q12HcD) in normal and tumor B lymphocytes. Strikingly, in normal B cells, we showed that the 1q12HcD dynamically organizes to the nuclear periphery in response to B-cell receptor engagement. Specifically, we observed an almost twofold increase in 1q12Hc domains at the extreme nuclear periphery in activated versus resting B lymphocytes. Remarkably, 1q12Hc organization was noticeably altered in tumor cells that showed structural alterations of 1q12; the 1q12Hc domains were significantly displaced from the extreme nuclear periphery compared to normal activated B lymphocytes (P > 0.0001), although overall peripheral localization was maintained. In a case in which there was a translocation of IGL enhancer to 1q, the altered nuclear positioning of the 1q12HcD was even more pronounced (5% of the 1q12Hc domains at the nuclear periphery compared to 20% in other lymphoma lines), and we were able to mimic this effect in two additional B-cell tumor lines by treatment with trichostatin A, a histone deacetylase (HDAC) inhibitor. Taken together, these results point to the 1q12HcD having a specific, nonrandom, and regulated peripheral organization in B lymphocytes. This organization is significantly disrupted in lymphoma cells harboring 1q rearrangements. © 2005 Wiley-Liss, Inc.

International audience; Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (wt) P. aeruginosa. wt Y. enterocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN ( Delta HOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of Delta HOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopB/D proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon.

The growth factor-independent erythroleukemic cell line ERY-1 was established from the peripheral blood of a 87-year-old woman with chronic myeloid leukemia (CML) in the acute phase. Immunophenotyping showed that fresh leukemic cells were positive for CD13, CD33, CD36 and CD235a (glycophorin A), a phenotype compatible with that of erythroblastic cells. Cytogenetic and fluorescence in situ hybridization (FISH) analysis demonstrated classical t(9;22)(q34;q11) chromosomic translocation associated with a duplication of the BCR-ABL fusion gene. Other cytogenetic abnormalities were detected in all analyzed mitosis, the most frequent being a trisomy of chromosome 8. The established ERY-1 cell line retains these immunophenotypic and cytogenetic features, and light and electron microscopy confirmed the relatively mature erythroblastic phenotype of the cells. In addition, ERY-1 cell line expressed beta-globin mRNA and a non-phosphorylable form of the erythropoietin receptor, even in presence of erythropoietin. Of note, the proliferation of ERY-1 cells was inhibited by TGFbeta1 or STI-571 (Gleevec), without significant induction of further differentiation. In conclusion, ERY-1 is a new growth factor-independent human erythroleukemic cell line with a relatively mature phenotype that may be useful to study the molecular events involved in erythroblastic differentiation.

Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)-1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All-1 −/− mouse embryonic stem cells and reduced in All-1 +/− cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL-1 and could be considered as important tools for the study of the early leukemic cell phenotype.

Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients.

International audience; Cryptic unbalanced subtelomeric rearrangements are known to cause a significant proportion of idiopathic mental retardation in childhood. Because of the limited sensitivity of routine analyses, the cytogenetic detection of such rearrangements requires molecular techniques, namely FISH and comparative genomic hybridisation (CGH). An alternative approach consists in using genetic markers to detect segmental aneusomy. Here, we describe a new strategy based upon automated fluorescent genotyping to search for non mendelian segregation of telomeric microsatellites. A total of 29 individuals belonging to 24 unrelated families were screened and three abnormal patterns of segregation were detected (two rearrangements and one parental disomy). This study gives strong support to the view that cryptic telomeric rearrangements significantly contribute to idiopathic mental retardation and demonstrates that fluorescent genotyping is a very sensitive and cost-effective method to detect deletions, duplications and uniparental disomies.

Mitochondria, though containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. If the mechanisms of pre-protein import are well understood, the import of nuclear-coded RNAs into mitochondria was investigated to a much lesser extent. This targeting, if not universal, is widely spread among species. The origin and the mechanisms of RNA import seem to differ from one system to another and striking differences are observed even in closely related species. We describe data concerning the various experimental systems of studying RNA import with emphasis on the model of the yeast Saccharomyces cerevisiae, which was studied in our laboratory. We compare various requirements of RNA import into mitochondria in different species and demonstrate that this pathway can be transferred from yeast to human cells, in which tRNAs normally are not imported. We speculate on the possibility to use RNA import for biomedical purposes.

International audience; The promyelocyte (PML)-retinoic acid receptor alpha (RARalpha) fusion gene results from a t(15;17) chromosome translocation in acute promyelocytic leukaemia. We have analysed the oncogenic potential of the human fusion PML-RARalpha product in chicken using retrovirus vectors. We show that PML-RARalpha transforms very early haematopoietic progenitor cells in vitro and induces acute leukaemias. Neither PML nor RARalpha domains alone achieve such a transformation. The PML-RARalpha viruses recovered from the transformed cells carry two point mutations in the PML domain, one of which alters both the pattern of intracellular localization of the fusion protein and its functional interference with AP-1, thus defining an essential domain in PML for oncogenic transformation.

International audience; The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

International audience; Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.

International audience; We report here that the fusion of PML, a nuclear protein defined by the t(15;17) chromosomal translocation in acute promyelocytic leukemia, with retinoic acid receptor alpha (RAR alpha) changes the RAR alpha from a retinoic acid (RA)-dependent inhibitor to a RA-dependent activator of AP-1 transcriptional activity. The PML-RAR alpha chimera cooperates with c-Jun and, strikingly, with c-Fos to stimulate the transcription of both synthetic and natural reporter genes containing an AP-1 site. Stimulation is dependent on the concentration of RA and its dose-response curve is comparable to that for activation by RAR alpha of transcription on RA-responsive genes. Further, in the absence of RA, a circumstance in which RAR alpha has no effect on AP-1 activity, PML-RAR alpha is an inhibitor. Deletion of the dimerization, transactivation, or DNA-binding domains of c-Jun and removal of the PML dimerization domain in the PML-RAR alpha hybrid abrogates their transcriptional cooperatively. In view of the association between AP-1 activity and hemopoietic differentiation, we suggest that these properties of PML-RAR alpha could contribute to the leukemic phenotype and its response to RA.

The t(15;17) translocation is specifically observed in patients with promyelocytic leukemia (AML3). The chromosomal rearrangement juxtaposes the retinoic acid receptor alpha (RAR alpha) and PML genes, resulting in PML/RAR alpha fusion transcripts. Our previous studies have shown that a polymerase chain reaction (PCR) amplification product could be obtained from the cDNA of the NB4 promyelocytic cell line from which the chimaeric PML/RAR alpha was cloned. We report here that in all 14 AML3 patients tested, reverse transcriptase-PCR (RT-PCR) allows the detection of three specific fusion products. In eight patients, one amplification product was detected corresponding to the previously described abnormal fusion. Five patients displayed a different amplified fragment corresponding to a different fusion point. One other patient always showed a third different-sized product. The different types of fusion transcripts amplified were correlated to the size of the abnormal RAR alpha transcripts detected in these patients by Northern analysis, but did not prove determinant for either the phenotypic features or the retinoic acid responsiveness in AML3 cells in this group of patients. The consistent identification by RT-PCR of the fusion of the PML and RAR alpha genes in AML3 patients suggest that this method will provide a useful tool for the diagnosis and detection of minimal residual disease in these patients.

International audience; We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RAR alpha mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RAR alpha, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RAR alpha progenitor. Identical PML-RAR alpha fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.

International audience; Retinoic acid is a vitamin A derivative with striking effects on development and cell differentiation. Several nuclear retinoic acid receptors (RARs), acting as ligand-inducible transcription factors, have been characterized and indirect evidence suggests that they have distinct roles. One of the most intriguing properties of retinoic acid is its ability to induce in vivo differentiation of acute promyelocytic leukaemia (APL) cells into mature granulocytes, leading to morphological complete remissions. Because the RAR alpha gene maps to chromosome 17q21 (ref. 14), close to the t(15;17) (q21-q11-22) translocation specifically associated with APL, we analysed RAR alpha gene structure and expression in APL cells. We report here that, in one APL-derived cell line, the RAR alpha gene has been translocated to a locus, myl, on chromosome 15, resulting in the synthesis of a myl/RAR alpha fusion messenger RNA. Using two probes located on either side of the cloned breakpoint, we have found genomic rearrangements of one or other locus in six patients out of eight, demonstrating that the RAR alpha and/or myl genes are frequently rearranged in APL and the breakpoints are clustered. These findings strongly implicate retinoic acid receptor alpha in leukaemogenesis.

International audience; A repeated DNA element (STIR) interspersed in Xp22.3 and on the Y chromosome has been used as a tag to isolate seven single-copy probes from the human sex chromosomes. The seven probes detect X-specific loci located in Xp22.3. Using a panel of X-chromosomal deletions from X-Y interchange sex reversals (XX males and XY females), these X-specific loci and some additional ones were mapped to four contiguous intervals of Xp22.3, proximal to the pseudoautosomal region and distal to STS. The construction of this deletion map of the terminal part of the human X chromosome can serve as a starting point for a long-range physical map of Xp22.3 and for a more accurate mapping of genetic diseases located in Xp22.3.