Showing papers on "Chromosome breakage published in 2012"

DNA breaks and chromosome pulverization from errors in mitosis

Abstract: The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for 'chromothripsis' in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.

Topoisomerase I poisoning results in PARP-mediated replication fork reversal

Abstract: Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.

Chromothripsis and cancer: causes and consequences of chromosome shattering

Abstract: Genomic alterations that lead to oncogene activation and tumour suppressor loss are important driving forces for cancer development. Although these changes can accumulate progressively during cancer evolution, recent studies have revealed that many cancer cells harbour chromosomes bearing tens to hundreds of clustered genome rearrangements. In this Review, we describe how this striking phenomenon, termed chromothripsis, is likely to arise through chromosome breakage and inaccurate reassembly. We also discuss the potential diagnostic, prognostic and therapeutic implications of chromothripsis in cancer.

Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements

Abstract: Next-generation sequencing of DNA from human tumors or individuals with developmental abnormalities has led to the discovery of a process we term chromoanagenesis, in which large numbers of complex rearrangements occur at one or a few chromosomal loci in a single catastrophic event. Two mechanisms underlie these rearrangements, both of which can be facilitated by a mitotic chromosome segregation error to produce a micronucleus containing the chromosome to undergo rearrangement. In the first, chromosome shattering (chromothripsis) is produced by mitotic entry before completion of DNA replication within the micronucleus, with a failure to disassemble the micronuclear envelope encapsulating the chromosomal fragments for random reassembly in the subsequent interphase. Alternatively, locally defective DNA replication initiates serial, microhomology-mediated template switching (chromoanasynthesis) that produces local rearrangements with altered gene copy numbers. Complex rearrangements are present in a broad spectrum of tumors and in individuals with congenital or developmental defects, highlighting the impact of chromoanagenesis on human disease.

Chromosomal instability and cancer: a complex relationship with therapeutic potential

Abstract: Chromosomal instability (CIN) is a hallmark of human neoplasms. Despite its widespread prevalence, knowledge of the mechanisms and contributions of CIN in cancer has been elusive. It is now evident that the role of CIN in tumor initiation and growth is more complex than previously thought. Furthermore, distinguishing CIN, which consists of elevated rates of chromosome missegregation, from aneuploidy, which is a state of abnormal chromosome number, is crucial to understanding their respective contributions in cancer. Collectively, experimental evidence suggests that CIN enables tumor adaptation by allowing tumors to constantly sample the aneuploid fitness landscape. This complex relationship, together with the potential to pharmacologically influence chromosome missegregation frequencies in cancer cells, offers previously unrecognized means to limit tumor growth and its response to therapy.

R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants

Abstract: Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing ~25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP–chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.

FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair

Abstract: Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.

A genome-wide analysis of common fragile sites: What features determine chromosomal instability in the human genome?

Abstract: Chromosomal common fragile sites (CFSs) are unstable genomic regions that break under replication stress and are involved in structural variation. They frequently are sites of chromosomal rearrangements in cancer and of viral integration. However, CFSs are undercharacterized at the molecular level and thus difficult to predict computationally. Newly available genome-wide profiling studies provide us with an unprecedented opportunity to associate CFSs with features of their local genomic contexts. Here, we contrasted the genomic landscape of cytogenetically defined aphidicolin-induced CFSs (aCFSs) to that of nonfragile sites, using multiple logistic regression. We also analyzed aCFS breakage frequencies as a function of their genomic landscape, using standard multiple regression. We show that local genomic features are effective predictors both of regions harboring aCFSs (explaining ∼77% of the deviance in logistic regression models) and of aCFS breakage frequencies (explaining ∼45% of the variance in standard regression models). In our optimal models (having highest explanatory power), aCFSs are predominantly located in G-negative chromosomal bands and away from centromeres, are enriched in Alu repeats, and have high DNA flexibility. In alternative models, CpG island density, transcription start site density, H3K4me1 coverage, and mononucleotide microsatellite coverage are significant predictors. Also, aCFSs have high fragility when colocated with evolutionarily conserved chromosomal breakpoints. Our models are predictive of the fragility of aCFSs mapped at a higher resolution. Importantly, the genomic features we identified here as significant predictors of fragility allow us to draw valuable inferences on the molecular mechanisms underlying aCFSs.

Selenium and its' role in the maintenance of genomic stability.

Abstract: Selenium (Se) is an essential micronutrient for humans, acting as a component of the unusual amino acids, selenocysteine (Se-Cys) and selenomethionine (Se-Met). Where Se levels are low, the cell cannot synthesise selenoproteins, although some selenoproteins and some tissues are prioritised over others. Characterised functions of known selenoproteins, include selenium transport (selenoprotein P), antioxidant/redox properties (glutathione peroxidases (GPxs), thioredoxin reductases and selenoprotein P) and anti-inflammatory properties (selenoprotein S and GPx4). Various forms of Se are consumed as part of a normal diet, or as a dietary supplement. Supplementation of tissue culture media, animal or human diets with moderate levels of certain Se compounds may protect against the formation of DNA adducts, DNA or chromosome breakage, and chromosome gain or loss. Protective effects have also been shown on mitochondrial DNA, and on telomere length and function. Some of the effects of Se compounds on gene expression may relate to modulation of DNA methylation or inhibition of histone deacetylation. Despite a large number of positive effects of selenium and selenoproteins in various model systems, there have now been some human clinical trials that have shown adverse effects of Se supplementation, according to various endpoints. Too much Se is as harmful as too little, with animal models showing a "U"-shaped efficacy curve. Current recommended daily allowances differ among countries, but are generally based on the amount of Se necessary to saturate GPx enzymes. However, increasing evidence suggests that other enzymes may be more important than GPx for Se action, that optimal levels may depend upon the form of Se being ingested, and vary according to genotype. New paradigms, possibly involving nutrigenomic tools, will be necessary to optimise the forms and levels of Se desirable for maximum protection of genomic stability in all humans.

Recurrent Rearrangement of the PHF1 Gene in Ossifying Fibromyxoid Tumors

Abstract: Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor of unknown lineage. Although most cases are histologically and clinically benign, some show malignant morphological features and local recurrences are not uncommon; a few may even metastasize. In the present study, cytogenetic analysis identified different structural rearrangements of chromosome band 6p21 in tumor cells from three cases of OFMT, including one with typical, one with atypical, and one with malignant morphological features. Mapping of the 6p21 breakpoint by fluorescence in situ hybridization (FISH) indicated that the PHF1 gene was rearranged in all three cases. Further FISH, 5'-rapid amplification of cDNA ends, and RT-PCR analyses disclosed an EP400/PHF1 fusion transcript in one of the cases. Interphase FISH on tumor sections from 13 additional cases of OFMT showed rearrangement of the PHF1 locus in four of four typical, two of three atypical, and one of six malignant lesions. Thus, the PHF1 gene, previously shown to be the 3'-partner of fusion genes in endometrial stromal tumors, is also recurrently involved in the pathogenesis of OFMTs, irrespective of whether they are diagnosed as typical, atypical, or malignant lesions. The PHF1 protein interacts with the polycomb-repressive complex 2 (PRC2), which, in turn, regulates the expression of a variety of developmental genes. Thus, the results indicate that deregulation of PRC2 target genes is crucial for OFMT development.

Mussel micronucleus cytome assay

Abstract: The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).

Exome sequencing reveals a novel Fanconi group defined by XRCC2 mutation

Abstract: Background Fanconi anaemia (FA) is a group of disorders characterised by progressive bone marrow failure and a characteristic but variable craniofacial and skeletal involvement. Recessive mutations in any of 15 genes linked to FA lead to the pathognomonic increased susceptibility to double-strand DNA breaks. Methods Autozygome and exome analysis of a patient with classic FA phenotype Results The authors identified a novel truncating mutation in XRCC2. Consistent with the proposed causal link to FA, this gene is an essential non-redundant component of the RAD51 family of homologous repair proteins and its deficiency in a murine model has been shown to lead to a highly similar phenotype to that of this patient both at the cellular and organismal level. Conclusion This study implicates XRCC2 in the pathogenesis of FA and calls for further investigation of the potential contribution of XRCC2 mutations to the overall mutational load of FA.

An improved approach for accurate and efficient calling of structural variations with low-coverage sequence data

Abstract: Background Recent advances in sequencing technologies make it possible to comprehensively study structural variations (SVs) using sequence data of large-scale populations. Currently, more efforts have been taken to develop methods that call SVs with exact breakpoints. Among these approaches, split-read mapping methods can be applied on low-coverage sequence data. With increasing amount of data generated, more efficient split-read mapping methods are still needed. Also, since sequence errors can not be avoided for the current sequencing technologies, more accurate split-read mapping methods are still needed to better handle sequence errors.

A tandem segmental duplication (TSD) in green revolution gene Rht-D1b region underlies plant height variation

Abstract: • Rht-D1c (Rht10) carried by Chinese wheat (Triticum aestivum) line Aibian 1 is an allele at the Rht-D1 locus. Among the Rht-1 alleles, little is known about Rht-D1c although it determines an extreme dwarf phenotype in wheat. • Here, we cloned and functionally characterized Rht-D1c using a combination of Southern blotting, target region sequencing, gene expression analysis and transgenic experiments. • We found that the Rht-D1c allele was generated through a tandem segmental duplication (TSD) of a > 1 Mb region, resulting in two copies of the Rht-D1b. Two copies of Rht-D1b in the TSD were three-fold more effective in reducing plant height than a single copy, and transformation with a segment containing the tandemly duplicated copy of Rht-D1b resulted in the same level of reduction of plant height as the original copy in Aibian 1. • Our results suggest that changes in gene copy number are one of the important sources of genetic diversity and some of these changes could be directly associated with important traits in crops.

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

Abstract: The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon.

Abstract: The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25°C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F6∶7 recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F2 population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.

Barbara McClintock and the discovery of jumping genes

Abstract: For much of the 20th century, genes were considered to be stable entities arranged in an orderly linear pattern on chromosomes, like beads on a string (1). In the late 1940s, Barbara McClintock challenged existing concepts of what genes were capable of when she discovered that some genes could be mobile. Her studies of chromosome breakage in maize led her to discover a chromosome-breaking locus that could change its position within a chromosome. McClintock went on to discover other such mobile elements, now known as transposons. She also found that depending on where they inserted into a chromosome these mobile elements could reversibly alter the expression of other genes. She summarized her data on the first transposable elements she discovered, Ac and Ds , in a 1950 PNAS Classic Article, “The origin and behavior of mutable loci in maize” (2). Although their existence was accepted relatively soon after by maize geneticists, the widespread nature of mobile genetic elements and the implications of McClintock’s discovery took decades to be widely recognized. Barbara McClintock at her laboratory desk, 1971. By the 1970s the great strides made in molecular biology led to the discovery of transposons in other organisms, starting with viruses and bacteria. We now know that transposons constitute more than 65% of our genomes and approximately 85% of the maize genome. “Transposons are astonishingly abundant, comprising a majority of the DNA in some species,” said Nina Fedoroff, a professor at Penn State University and King Abdullah University of Science and Technology and author of the PNAS Classic Perspective on McClintock’s article, “McClintock’s challenge in the 21st century” (3). Confirmation that transposons were widespread among eukaryotes eventually led to the wider appreciation of her original discovery. McClintock received a number of prestigious awards, including the 1970 National Medal of Science and culminating …

The cell adhesion gene PVRL3 is associated with congenital ocular defects.

Abstract: We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell–cell adhesion mechanism in human ocular development.

Chromosome Damage in Human Cells by γ Rays, α Particles and Heavy Ions: Track Interactions in Basic Dose-Response Relationships

Abstract: We irradiated normal human lymphocytes and fibroblasts with 137Cs γ rays, 3.5 MeV α particles and 1 GeV/amu 56Fe ions and measured the subsequent formation of chromosome-type aberrations by mFISH at the first mitosis following irradiation. This was done for the purposes of characterizing the shape of dose-response relationships and determining the frequency distribution of various aberration types with respect to the parameters of dose, radiation quality and cell type. Salient results and conclusions include the following. For low-LET γ rays, lymphocytes showed a more robust dose response for overall damage and a higher degree of upward curvature compared to fibroblasts. For both sources of high-LET radiation, and for both cell types, the response for simple and complex exchanges was linear with dose. Independent of all three parameters considered, the most likely damage outcome was the formation of a simple exchange event involving two breaks. However, in terms of the breakpoints making up exchange event...

A novel deletion in ZBTB24 in a Lebanese family with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2.

Abstract: The immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by targeted chromosome breakage, directly related to a genomic methylation defect. It manifests with phenotypic and clinical variability, with the most consistent features being developmental delay, facial anomalies, cytogenetic defects and immunodeficiency with a reduction in serum immunoglobulin levels. From the molecular point of view, ICF syndrome was always divided into ICF type I (ICF1) and ICF type 2 (ICF2). Mutations in DNMT3B gene are responsible for ICF1, while mutations in ZBTB24 have been reported to be responsible for ICF2. In this study, we describe a Lebanese family with three ICF2 affected brothers. Sanger sequencing of the coding sequence of ZBTB24 gene was conducted and revealed a novel deletion: c.396_397delTA (p.His132Glnfs*19), resulting in a loss-of-function of the corresponding protein. ZBTB24 belongs to a large family of transcriptional factors and may be involved in DNA methylation of juxtacentromeric DNA. Detailed molecular and functional studies of the ZBTB24 and DNMT3B genes are needed to understand the pathophysiology of ICF syndrome.

Evaluation of micronuclei in tobacco users: A study in Punjabi population.

Abstract: Introduction: The assessment of micronuclei in exfoliated cells is a promising tool for the study of epithelial carcinogens and can be used to detect chromosome breakage or mitotic interference, thought to be relevant to carcinogenesis. Aim: The present study aimed to detect micronuclei in exfoliated oral mucosal cells in individuals using various tobacco forms from the last 5 years. Materials and Methods: A total of 75 healthy male subjects (25 smokeless tobacco users, 25 smokers, and 25 non-tobacco users) were selected for the study. Smears were obtained with moistened wooden spatula from buccal mucosa and fixed with 95% alcohol. All the cytologic smears were stained by Papanicolaou technique. From each slide, ~1000 cells were examined under the 400× magnification and where micronucleated (MN) cells were located, they were examined under the 1000× magnification. Result: MN cells were found to be significantly higher in smokeless tobacco users than in smokers and controls. Conclusion: A positive correlation is found between increased micronucleus frequency and tobacco-using habits. So micronucleus assay can be used as a biomarker of genotoxicity.

Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi.

Abstract: The subtelomeres of many protozoa are highly enriched in genes with roles in niche adaptation T cruzi trypomastigotes express surface proteins from Trans-Sialidase (TS) and Dispersed Gene Family-1 (DGF-1) superfamilies which are implicated in host cell invasion Single populations of T cruzi may express different antigenic forms of TSs Analysis of TS genes located at the telomeres suggests that chromosome ends could have been the sites where new TS variants were generated The aim of this study is to characterize telomeric and subtelomeric regions of T cruzi available in TriTrypDB and connect the sequences of telomeres to T cruzi working draft sequence We first identified contigs carrying the telomeric repeat (TTAGGG) Of 49 contigs identified, 45 have telomeric repeats at one end, whereas in four contigs the repeats are located internally All contigs display a conserved telomeric junction sequence adjacent to the hexamer repeats which represents a signature of T cruzi chromosome ends We found that 40 telomeric contigs are located on T cruzi chromosome-sized scaffolds In addition, we were able to map several telomeric ends to the chromosomal bands separated by pulsed-field gel electrophoresis The subtelomeric sequence structure varies widely, mainly as a result of large differences in the relative abundance and organization of genes encoding surface proteins (TS and DGF-1), retrotransposon hot spot genes (RHS), retrotransposon elements, RNA-helicase and N-acetyltransferase genes While the subtelomeric regions are enriched in pseudogenes, they also contain complete gene sequences matching both known and unknown expressed genes, indicating that these regions do not consist of nonfunctional DNA but are instead functional parts of the expressed genome The size of the subtelomeric regions varies from 5 to 182 kb; the smaller of these regions could have been generated by a recent chromosome breakage and telomere healing event The lack of synteny in the subtelomeric regions suggests that genes located in these regions are subject to recombination, which increases their variability, even among homologous chromosomes The presence of typical subtelomeric genes can increase the chance of homologous recombination mechanisms or microhomology- mediated end joining, which may use these regions for the pairing and recombination of free ends

The In Vitro Micronucleus Assay

Abstract: The in vitro micronucleus test detects genotoxic damage in interphase cells. The in vitro micronucleus test provides an alterative to the chromosome aberration test, and because the in vitro micronucleus test examines cells at interphase, the assessment of micronuclei can be scored faster, as the analysis of damage is thought to be less subjective and is more amenable to automation.Micronuclei may be the result of aneugenic (whole chromosome) or clastogenic (chromosome breakage) damage. This chapter provides methods for mononucleate and binucleate micronucleus tests and the addition of centromeric labelling and a non-disjunction assay to investigate any potential aneugenic mode of action.

Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer

Abstract: Introduction: Genomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex. Aim: We aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility. Methods: To this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects. Results: We demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common...

Shattered and stitched chromosomes-chromothripsis and chromoanasynthesis-manifestations of a new chromosome crisis?

Abstract: Chromothripsis (chromosome shattering) has been described as complex rearrangements affecting single chromosome(s) in one catastrophic event. The chromosomes would be "shattered" and "stitched together" during this event. This phenomenon is proposed to constitute the basis for complex chromosomal rearrangements seen in 2-3% of all cancers and in ∼ 25% of bone cancers. Here we discuss chromothripsis, the use of this term and the evidence presented to support a single catastrophic event that remodels the genome in one step. We discuss why care should be taken in using the term chromothripsis and what evidence is lacking to support its use while describing complex rearrangements.

Evidence for involvement of GNB1L in autism.

Abstract: Structural variations in the chromosome 22q11.2 region mediated by nonallelic homologous recombination result in 22q11.2 deletion (del22q11.2) and 22q11.2 duplication (dup22q11.2) syndromes. The majority of del22q11.2 cases have facial and cardiac malformations, immunologic impairments, specific cognitive profile and increased risk for schizophrenia and autism spectrum disorders (ASDs). The phenotype of dup22q11.2 is frequently without physical features but includes the spectrum of neurocognitive abnormalities. Although there is substantial evidence that haploinsufficiency for TBX1 plays a role in the physical features of del22q11.2, it is not known which gene(s) in the critical 1.5 Mb region are responsible for the observed spectrum of behavioral phenotypes. We identified an individual with a balanced translocation 46,XY,t(1;22)(p36.1;q11.2) and a behavioral phenotype characterized by cognitive impairment, autism, and schizophrenia in the absence of congenital malformations. Using somatic cell hybrids and comparative genomic hybridization (CGH) we mapped the chromosome-22 breakpoint within intron 7 of the GNB1L gene. Copy number evaluations and direct DNA sequencing of GNB1L in 271 schizophrenia and 513 autism cases revealed dup22q11.2 in two families with autism and private GNB1L missense variants in conserved residues in three families (P = 0.036). The identified missense variants affect residues in the WD40 repeat domains and are predicted to have deleterious effects on the protein. Prior studies provided evidence that GNB1L may have a role in schizophrenia. Our findings support involvement of GNB1L in ASDs as well. © 2011 Wiley Periodicals, Inc.

A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells

Abstract: Background: Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus During the last few decades, micronuclei (MNi) in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays Aim: To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, ie,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E) Materials and Methods: Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No of MNi/1000 cells) were compared using three different stains Results: Mean MNi frequency in cases was found to be 38 with Feulgen stain, 168 with PAP and 259 with H and E In controls, mean MNi frequency was 16 with Feulgen stain, 77 with PAP and 96 with H and E stain Statistically significant value (P Conclusion: Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains