Translocation capture sequencing: A method for high throughput mapping of chromosomal rearrangements

Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangements genome-wide in primary cells, and to discover recombination hotspots. Demonstrating its effectiveness, we successfully estimate the frequency of c-myc/IgH translocations in primary B cells, and identify hotspots of AID-mediated recombination. Furthermore. TC-Seq can be adapted to generate genome-wide rearrangement maps in any cell type and under any condition. (C) 2011 Elsevier B.V. All rights reserved.

Loss of heterozygosity induced by a chromosomal double-strand break

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Chromosomal aberrations in PARP−/− mice: Genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP−/− mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP−/−mice and immortalized PARP−/−fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP−/− cells stably transfected with PARP cDNA [PARP−/−(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP−/− cells, p53 expression was partially restored in PARP−/− (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP−/− mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.

The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.

Evidence for microsatellite instability in bilateral breast carcinomas

The molecular pathogenesis of various categories of breast cancer (BC) has been well described, but surprisingly few reports have appeared on analysis of somatic mutations in bilateral BC. We have performed a polymerase chain reaction (PCR)-driven investigation of chromosomal regions showing common loss of heterozygosity (LOH) in 23 cases (46 rumors) from patients diagnosed with bilateral BC, LOH was observed in 15/46 (33%) informative tumors for chromosome 1p, 5/32 (16%) for 5q, 12/44 (27%) for 11q, 15/40 (38%) for 13q and 4/24 (17%) for 17p. These values are within the range of interlaboratory variations reported fur unilateral BC, There was no strong evidence for concordance of LOH within the same patient for any of the chromosomal loci tested. Atypical for breast carcinomas, 7/46 (15%) turners accumulated a high frequency (ranging from 11 to 29%) of shortened dinucleotide CA repeats, implying microsatellite instability (MI). Further analysis with the highly informative BAT-26 marker allowed for the classification of two of these tumors as having a replication error positive (RER+/MSI-H) phenotype, whereas the remaining five carcinomas harbored so-called borderline MI. Thus an involvement of both RER+ and borderline MI appears to be a distinct feature of bilateral breast carcinomas compared to unilateral lesions. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.

Letter To The Editor: Atlantoaxial Instability And Chiari Malformation.

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Prediction of good glass formers in the Al-Ni-La and Al-Ni-Gd systems using topological instability and electronegativity

A new criterion has been recently proposed combining the topological instability (lambda criterion) and the average electronegativity difference (Delta e) among the elements of an alloy to predict and select new glass-forming compositions. In the present work, this criterion (lambda.Delta e) is applied to the Al-Ni-La and Al-Ni-Gd ternary systems and its predictability is validated using literature data for both systems and additionally, using own experimental data for the Al-La-Ni system. The compositions with a high lambda.Delta e value found in each ternary system exhibit a very good correlation with the glass-forming ability of different alloys as indicated by their supercooled liquid regions (Delta T(x)) and their critical casting thicknesses. In the case of the Al-La-Ni system, the alloy with the largest lambda.Delta e value, La(56)Al(26.5)Ni(17.5), exhibits the highest glass-forming ability verified for this system. Therefore, the combined lambda.Delta e criterion is a simple and efficient tool to select new glass-forming compositions in Al-Ni-RE systems. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3563099]

Gene Expression Profiles in Radiation Workers Occupationally Exposed to Ionizing Radiation

Ionizing radiation OR) imposes risks to human health and the environment. IR at low doses and low (lose rates has the potency to initiate carcinogenesis. Genotoxic environmental agents such as IR trigger a cascade of signal transduction pathways for cellular protection. In this study, using cDNA microarray technique, we monitored the gene expression profiles in lymphocytes derived from radiation-ex posed individuals (radiation workers). Physical dosimetry records on these patients indicated that the absorbed dose ranged from 0.696 to 39.088 mSv. Gene expression analysis revealed statistically significant transcriptional changes in a total of 78 genes (21 up-regulated and 57 clown-regulated) involved in several biological processes such as ubiquitin cycle (UHRF2 and PIAS1), DNA repair (LIG3, XPA, ERCC5, RAD52, DCLRE1C), cell cycle regulation/proliferation (RHOA, CABLES2, TGFB2, IL16), and stress response (GSTP1, PPP2R5A, DUSP22). Some of the genes that showed altered expression profiles in this study call be used as biomarkers for monitoring the chronic low level exposure in humans. Additionally, alterations in gene expression patterns observed in chronically exposed radiation workers reinforces the need for defining the effective radiation dose that causes immediate genetic damage as well as the long-term effects on genomic instability, including cancer.

Ring chromosome instability evaluation in six patients with autosomal rings

Ring chromosomes are often associated with abnormal phenotypes due to loss of genomic material and also because of ring instability at mitosis after sister chromatid exchange events. We investigated ring chromosome instability in six patients with ring chromosomes 4, 14, 15, and 18 by examining 48- and 72-h lymphocyte cultures at the first, second and subsequent cell divisions after bromodeoxyuridine incorporation. Although most cells from all patients showed only one monocentric ring chromosome, ring chromosome loss and secondary aberrations were observed both in 48-and 72-h lymphocyte cultures and in metaphase cells of the different cell generations. We found no clear-cut correlation between ring size and ring instability; we also did not find differences between apparently complete rings and rings with genetic material loss. The cytogenetic findings revealed secondary aberrations in all ring chromosome patients. We concluded that cells with ring chromosome instability can multiply and survive in vivo, and that they can influence the patient's phenotype.

Variability in Estrogen-Metabolizing Genes and Their Association with Genomic Instability in Untreated Breast Cancer Patients and Healthy Women

In the present study, we investigated the relationship between polymorphisms in the estrogen-metabolizing genes CYP17, CYP1B1, CYP1A1, and COMT and genomic instability in the peripheral blood lymphocytes of 62 BC patients and 62 controls considering that increased or prolonged exposure to estrogen can damage the DNA molecule and increase the genomic instability process in breast tissue. Our data demonstrated increased genomic instability in BC patients and that individuals with higher frequencies of MN exhibited higher risk to BC when belonging Val/Met genotype of the COMT gene. We also observed that CYP17 and CYP1A1 polymorphisms can modify the risk to BC depending on the menopause status. We can conclude that the genetic background in estrogen metabolism pathway can modulate chromosome damage in healthy controls and patients and thereby influence the risk to BC. These findings suggest the importance to ally biomarkers of susceptibility and effects to estimate risk groups.

Tetraploidization in Wilms tumor in an infant

Genetic instability is frequent in human cancer. Unscheduled tetraploidization can trigger cell transformation and tumorigenesis. We made a cytogenetic analysis by Giemsa-trypsin banding of a stage I, biphasic Wilms tumor diagnosed in a 10-month-old male. An evident karyotypic heterogeneity was found. Four different subclones of tumor cells were observed, with DNA content varying from diploid to near-tetraploid complements. The genetic events involved in the acquisition of aneuploidy in Wilms tumor remain unclear. We hypothesize that initial tetraploidization caused aberrant cell division, leading to abnormal chromosomal segregation, cell transformation and tumorigenesis.

Genome Size, Karyotype Polymorphism and Chromosomal Evolution in Trypanosoma cruzi

Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites. Methodology/Principal Findings: The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (Jose-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively. Conclusions: Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.