Spontaneous Chromosome Breakage Research Articles

Loss of CBX2 causes genomic instability and Wnt activation in high grade serous ovarian carcinoma cells.

High grade serous ovarian carcinoma (HGSOC) is lethal with insidious onset, rapid progression, poor prognosis, and limited treatment options. Polycomb repressor complexes (PRC) 1 and 2 are intimately involved in progression of many types of cancer including HGSOC. Unlike the consistent constitution of PRC2, PRC1 consists of diverse components whose clinical significance in HGSOC are not entirely clear. Here, prognosis-associated PRC1 components were identified through data-mining. CBX2 promoted proliferation and reduced apoptosis of HGSOC cell lines OVCAR4, OVCAR3, and CAOV3. Complete loss of CBX2 by CRISPR-cas9 editing (CBX2KO ) destabilized genome stability with increased spontaneous chromosomal breaks and tendency to polyploidy accompanied by disrupted cell cycle especially stalled G2/M transition and caused severe cell death. Wnt/β-catenin/LEF1/TCF7L1 was activated in surviving OVCAR4-CBX2KO clones to bypass the crisis caused by loss of CBX2. The relieve of TCF7L1 core-promoter region occupied by CBX2 might be one of the possible explanations to TCF7L1 increase in OVCAR4-CBX2KO clones. Subcutaneous tumor model further validated that depletion of CBX2 repressed HGSOC cell line derived tumor growth. High immunohistochemistry score of CBX2 in primary ovarian cancer tissue associated with advanced clinical stage (p = 0.033), poor overall survival (HR = 3.056, 95% CI: 1.024-9.123), and progression free survival (HR = 4.455, 95% CI: 1.513-13.118) in HGSOC. Overall, our results suggested that CBX2 was a promising prognostic factor and therapeutic target in HGSOC.

Replication stress impairs chromosome segregation and preimplantation development in human embryos

Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.

Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.

Influence of Polymorphisms of DNA Repair and GST Genes on Genotoxic Damage and Mutagen Sensitivity in Workers Occupationally Exposed to Very Low Doses of Ionizing Radiation

The study investigated the influence of genetic polymorphisms of the enzymes for DNA repair and detoxification of reactive intermediates on spontaneous and bleomycin-induced (BLM) genotoxic damage in 43 workers exposed to very low doses of ionizing radiation (IR) (mean cumulative dose 5.31 mSv) and 43 subjects with no occupational exposure to IR (controls). In all the subjects examined, the frequency of chromosome aberrations (CAs) and micronuclei (MN), both spontaneous and BLM-induced, the Comet assay parameters (tail intensity), the genotypic variants of the DNA repair enzymes XRCC1 (Arg194Trp, Arg280His, Arg399Gln), XRCC3 (Thr241Met), XPD (Lys751Gln), and of the detoxification enzymes GSTM1 and GSTT1 (null genotype) and BLMH (A1450G) were determined. Among the biomarkers considered, only the frequency of total CAs (p < 0.05), and in particular of chromosome breaks (p < 0.01), was found to be significantly higher in the exposed workers than the controls. The frequency of spontaneous MN was higher in subjects with at least one allelic variant in XRCC1 than in carriers of the wild-type, but again only in exposed workers (p = 0.046). Linear regression analysis showed a positive dependency of the frequency of spontaneous chromosome breaks on occupational exposure, and a dependency of the frequency of BLM-induced MN negative on occupational exposure and positive on alcohol consumption and the null GSTM1 genotype. In conclusion, the frequency of chromosome breaks seems to be a useful cytogenetic biomarker for exposure to very low doses of IR, while only the combined effect of different gene variants or genetic, occupational, and lifestyle habits factors seems to be able to modulate the genotoxic effect of very low doses of IR.

Molecular insights into replication‐associated genome instabilities caused by CST deficiency

The RPA‐like ssDNA binding protein complex CTC1/STN1/TEN1 (CST) has recently been implicated in promoting genome stability under replications stress. Deficiency in CST genes cause the Coats plus syndrome, a complex genetic disorder affecting multiple systems including brain, retina, bone, gastrointestine, and other parts of the body. Yet the precise role of CST in countering replications stress is unclear. Recently we have reported that CST is enriched at GC‐rich and repetitive fragile sequences genome‐wide in response to hydroxyurea (HU)‐induced replication stress (Chastain et al, 2016, Cell Rep 16, 1300–1314). CST deficiency leads to spontaneous chromosome breakage and high‐level chromosome fragmentation that can be further exacerbated by replication stress. Mechanistically, we have found that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association between CST with RAD51 in an ATR‐dependent and DNA‐independent manner. Strikingly, CST deficiency diminishes HU‐induced RAD51 foci formation and reduces RAD51 recruitment to GC‐rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC‐rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. In the present research, we characterize eleven reported disease‐causing CTC1 missense mutations in maintaining genome stability. Our results show that these mutations induce spontaneous chromosome breakage that leads to global genome instabilities. These mutations abolish or reduce CST interaction with RAD51, disrupt RAD51 foci formation, and/or diminish binding to GC‐rich genomic fragile sites under replication stress. Furthermore, CTC1 mutations limit cell proliferation under unchallenged condition and significantly reduce clonal viability under replication stress. Our findings thus provide molecular evidence linking replication‐associated genomic defects with Coats plus disease pathology.Support or Funding InformationResearch is supported by NIH R01GM112864.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pathogenic CTC1 mutations cause global genome instabilities under replication stress.

Coats plus syndrome is a complex genetic disorder that can be caused by mutations in genes encoding the CTC1–STN1–TEN1 (CST) complex, a conserved single-stranded DNA binding protein complex. Studies have demonstrated that mutations identified in Coats plus patients are defective in telomere maintenance, and concluded that Coats plus may be caused by telomere dysfunction. Recent studies have established that CST also plays an important role in countering replication stress and protecting the stability of genomic fragile sites. However, it is unclear whether instabilities at genomic regions may promote Coats plus development. Here, we characterize eleven reported disease-causing CTC1 missense and small deletion mutations in maintaining genome stability. Our results show that these mutations induce spontaneous chromosome breakage and severe chromosome fragmentation that are further elevated by replication stress, leading to global genome instabilities. These mutations abolish or reduce CST interaction with RAD51, disrupt RAD51 foci formation, and/or diminish binding to GC-rich genomic fragile sites under replication stress. Furthermore, CTC1 mutations limit cell proliferation under unstressed condition and significantly reduce clonal viability under replication stress. Results also suggest that the aa 600–989 region of CTC1 contains a RAD51-interacting domain. Our findings thus provide molecular evidence linking replication-associated genomic defects with CP disease pathology.

Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice

AbstractFanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2−/− mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2−/− mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2−/−Trp53+/− mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient–derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.

FANCN Hypomorphic Mutation Retains BRCA1 Binding Domain

Introduction: Fanconi anemia (FA) is a recessive genetic disease that can result in bone marrow failure, morphological defects and cancer predisposition. Patients with mutations in the FA gene, FANCN/PALB2 (partner and localizer of BRCA2) typically have more severe disease with earlier onset of cancer and bone marrow failure. FANCN is known to promote homologous recombination (HR) and contains an N-terminal BRCA1 binding domain, and a C-terminal BRCA2 binding domain. To further investigate the function of FANCN, we used recombinant adeno-associated virus (rAAV)-mediated gene targeting techniques to make a conditional null FANCN cell line. We targeted early exons 2 and 3 and found that removal of the conditional allele resulted in spontaneous chromosomal breaks and rearrangements that ultimately lead to chromosomal catastrophe within 48 hours. Due to the essential function of FANCN in genome stability, we were curious how FANCN patients can survive with biallelic loss of function mutations in FANCN.Methods: A hypomorphic FANCN cell line was created using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9)-mediated gene targeting of FANCN in exon 5. In addition, we complemented the FANCN conditional null cell line with four different FANCN cDNAs containing patient mutations with premature stop codons that completely eliminate or disrupt the N-terminal BRCA2 binding domain. These FANCN mutant cell lines have been investigated for expression of truncated protein and will be tested in their ability to bind and form foci with BRCA1 and BRCA2. Finally, these FANCN mutant cell lines will be evaluated in their sensitivity to mitomycin C (MMC), PARP inhibitors, replication stress, and in their ability to repair DNA.Results: We have determined that FANCN is essential for maintaining genomic stability and cellular viability. However, contrary to the lethality of deleting exons 2 and 3, the biallelic frameshift mutations in exon 5 resulted in a viable FANCN mutant cell line. These hypomorphic FANCN exon 5 frameshift mutations truncate the C-terminal BRCA2 binding domain, and this cell line has a growth defect and MMC sensitivity. Furthermore, we have demonstrated that the patient mutation complemented cell lines are expressing truncated FANCN proteins that will be functionally characterized.Conclusions: We are beginning to unveil important and separate functional domains of FANCN. For example, the FANCN N-terminus with the BRCA1 binding domain serves an essential function in viability and genomic stability, whereas the FANCN C-terminus with the BRCA2 binding domain servers an essential role in cell proliferation and MMC resistance. This research is expected to continue to advance our mechanistic understanding of FANCN and how these FANCN patient mutations result in disease. In addition, these novel FANCN cell lines provide a means for screening potential new therapeutics for the treatment of Fanconi anemia. DisclosuresHendrickson:Horizon Discovery , Humanetics and Intellia Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: licensing fees, Research Funding.

Phenotypes and genotypes of the chromosomal instability syndromes.

As defined initially, chromosome instability syndromes (CIS) are a group of inherited conditions transmitted in autosomal recessive pattern characterised with both mental and physical development delay generally. They are also with other medical complications in individuals with CIS commonly including different degree of dysmorphics, organs/systems dys-function and high risk of cancer predisposition. Chromosomal breakage from CIS can be seen either in spontaneous breakage around 10-15% observed in Fanconi anemia or induced by clastogenic agents such as mitomycin (MMC), diepoxybutane (DEB). The spontaneous chromosome breakage is less common but it correlates with patient clinical severity. Relative high rates of some types of CIS can occur in certain ethnic groups. Individuals with CIS are commonly in childhood and these disorders are often lethal. Diagnosis is complicated usually because the symptoms presented from individuals with CIS may be varied and complex. Advances in molecular level have identified genes responsible for such group diseases/disorders demonstrated that CIS are characterized by the genome instability, defect in DNA repair mechanisms. Latest advances in high-throughput technologies have been increasing sequencing capabilities to facilitate more accurate data for such syndrome researches. CIS are the typical rare diseases and becoming more challenges in pediatrics clinic. In the last two decades, there were no many articles to review and analysis CIS together to comparing their phenotypes and genotypes. In this article, the similarity and differences of the phenotypes and genotypes of CIS were reviewed to understanding the whole profiles of CIS to assist laboratory genetic diagnostic services in CIS and for the confirmation from the clinical referrals.

Fanconi Anemia, Complementation Group D1 Caused by Biallelic Mutations of BRCA2 Gene--Case Report

Fanconi anemia is a rare autosomal recessive disorder, clinically and genetically heterogeneous, characterized by typical clinical features, such as short stature, microcephaly, skeletal abnormalities, abnormal skin pigmentations, developmental delay and congenital heart, kidney anomalies etc. Pancytopenia leading to bone marrow failure occurs in the first decade. Patients with Fanconi anemia have a high risk of hematologic malignancies and solid tumors. The diagnosis of Fanconi anemia is based on cytogenetic testing for increased rates of spontaneous chromosomal breakage and increased sensitivity to diepoxybutane or mitomycin C. Fanconi anemia is a heterogeneous disorder, at least 15 complementation groups are described, and 15 genes in which mutations are responsible for all of the 15 Fanconi anemia complementation groups have been identified. Unlike other Fanconi anemia complementation groups, for complementation group D1 (FANCD1), the bone marrow failure is not a typical feature, but early-onset leukemia and specific solid tumors, most often medulloblastoma and Wilms tumor, are typical for this complementation group.

REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

DNA polymerase zeta (pol ζ) is exceptionally important for controlling mutagenesis and genetic instability. REV3L comprises the catalytic subunit, while REV7 (MAD2L2) is considered an accessory subunit. However, it has not been established that the role of REV7 in DNA damage tolerance is necessarily connected with mammalian pol ζ, and there is accumulating evidence that REV7 and REV3L have independent functions. Analysis of pol ζ has been hampered by difficulties in expression of REV3L in mammalian cells, and lack of a functional complementation system. Here, we report that REV7 interacts with full-length REV3L in vivo and we identify a new conserved REV7 interaction site in human REV3L (residues 1993–2003), distinct from the known binding site (residues 1877–1887). Mutation of both REV7-binding sites eliminates the REV3L–REV7 interaction. In vivo complementation shows that both REV7-binding sites in REV3L are necessary for preventing spontaneous chromosome breaks and conferring resistance to UV radiation and cisplatin. This demonstrates a damage-specific function of REV7 in pol ζ, in contrast to the distinct roles of REV3L and REV7 in primary cell viability and embryogenesis.

The SNM1B/APOLLO DNA nuclease functions in resolution of replication stress and maintenance of common fragile site stability

SNM1B/Apollo is a DNA nuclease that has important functions in telomere maintenance and repair of DNA interstrand crosslinks (ICLs) within the Fanconi anemia (FA) pathway. SNM1B is required for efficient localization of key repair proteins, such as the FA protein, FANCD2, to sites of ICL damage and functions epistatically to FANCD2 in cellular survival to ICLs and homology-directed repair. The FA pathway is also activated in response to replication fork stalling. Here, we sought to determine the importance of SNM1B in cellular responses to stalled forks in the absence of a blocking lesion, such as ICLs. We found that depletion of SNM1B results in hypersensitivity to aphidicolin, a DNA polymerase inhibitor that causes replication stress. We observed that the SNM1B nuclease is required for efficient localization of the DNA repair proteins, FANCD2 and BRCA1, to subnuclear foci upon aphidicolin treatment, thereby indicating SNM1B facilitates direct repair of stalled forks. Consistent with a role for SNM1B subsequent to recognition of the lesion, we found that SNM1B is dispensable for upstream events, including activation of ATR-dependent signaling and localization of RPA, γH2AX and the MRE11/RAD50/NBS1 complex to aphidicolin-induced foci. We determined that a major consequence of SNM1B depletion is a marked increase in spontaneous and aphidicolin-induced chromosomal gaps and breaks, including breakage at common fragile sites. Thus, this study provides evidence that SNM1B functions in resolving replication stress and preventing accumulation of genomic damage.

Structure-Specific Endonucleases Xpf and Mus81 Play Overlapping but Essential Roles in DNA Repair by Homologous Recombination

DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here, we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR, as evidenced by the data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and to preventing early steps in HR by deleting XRCC3 suppressed the nonviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.

Current clinical management of Fanconi anemia

Fanconi anemia (FA) is a heterogeneous disease characterized by spontaneous chromosomal breaks and abnormal DNA repair. Major clinical problems in FA include congenital abnormalities, endocrinopathies, early onset bone marrow failure and increased risk of myelodysplastic syndrome, acute leukemia and solid tumors. To date, 15 different genes have been shown to cause FA, all of which have some role in DNA double-strand break repair. Very few strict genotype–phenotype associations have been identified and clinical manifestations vary widely from patient to patient, most likely due to modifier genes, environment and chance effects. Hematopoietic stem cell transplantation is the only proven cure for the hematopoietic manifestations of FA and aggressive lifelong surveillance for solid tumors is essential.

Heightened DNA damage response impairs hematopoiesis in Fanconi anemia

Since its first description by Guido Fanconi in 1927,1 Fanconi anemia (FA) has become one of the best characterized inherited bone marrow failure syndromes.2–3 Numerous studies over the years have documented its clinical and biological features. It is recognized as being clinically heterogeneous and patients can have a wide range of developmental abnormalities. Many patients develop progressive bone marrow failure and there is a high predisposition to cancer. FA cells characteristically display a high frequency of spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents4 such as mitomycin C and diepoxybutane. This FA cell hallmark has facilitated diagnosis in the clinic and scientific advances in the laboratory. FA is now known to be caused by biallelic mutations in one of 15 genes, the products of which interact in the FA/BRCA pathway in response to cellular stress and DNA damage to maintain genomic stability.5 FA can, therefore, be regarded as an inherited DNA repair disorder. Despite these advances, the precise pathophysiology of the bone marrow failure in FA has remained elusive. A recent international study by Ceccaldi and colleagues6 now provides new insights into the pathogenesis of bone marrow failure in FA. They analyzed bone marrow samples from a large number of FA patients and controls. By undertaking quantitative and qualitative assessment of hematopoietic function they established that FA patients have a significant impairment of hematopoiesis that begins early and is progressive throughout life. In view of the DNA repair defect that has been observed in FA cells, together with the observation that p53 plays an important role in the response to DNA damage, Ceccaldi and colleagues6 decided to analyze p53. They found strong basal and mitomycin C-induced p53 expression in primary cells from FA patients and in bone marrow cells of FA mouse (Fancg−/− and Fancd2−/−) models. To confirm that the p53 induction was directly related to the FA/BRCA pathway defect they knocked down FANCD2 (a key target of the FA pathway) in a human cell line (293T cells) and observed that p53, as well as p21, was induced in these cells. To mimic the response of FA cells to endogenous DNA damage, and to determine the link to the progressive cell impairment observed in patients, they exposed EBV-immortalized FA cells to interstrand cross-linking agents and studied the cellular response at several time points. These EBV-immortalized FA cells displayed the classic excess G2 arrest at 24 h; however, at later time points the G2 arrest resolved and the cells shifted to G0/G1 cell cycle arrest. Interestingly, in p53-silenced FA cells, p21 induction and G0/G1 accumulation were less marked. Complementary studies in FA mice showed that p53 impairs the hematopoietic and mitotic potential of Fanc-deficient murine hematopoietic stem and progenitor cells. Additional studies (including on bone marrow from young FA patients and fetal liver of FA patients) demonstrated constitutive activation of p53 and p21 as well as G0/G1 cell cycle arrest in hematopoietic progenitor cells in vivo. In conclusion, this study identifies an exacerbated p53/p21 DNA damage response as an important factor in the progressive impairment of hematopoiesis in FA; this begins prenatally and typically results in overt bone marrow failure in childhood. These new observations have important implications for the management of FA patients.

Nijmegen breakage syndrome (NBS)

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. Due to a founder mutation in the underlying NBN gene (c.657_661del5) the disease is encountered most frequently among Slavic populations. The principal clinical manifestations of the syndrome are: microcephaly, present at birth and progressive with age, dysmorphic facial features, mild growth retardation, mild-to-moderate intellectual disability, and, in females, hypergonadotropic hypogonadism. Combined cellular and humoral immunodeficiency with recurrent sinopulmonary infections, a strong predisposition to develop malignancies (predominantly of lymphoid origin) and radiosensitivity are other integral manifestations of the syndrome. The NBN gene codes for nibrin which, as part of a DNA repair complex, plays a critical nuclear role wherever double-stranded DNA ends occur, either physiologically or as a result of mutagenic exposure. Laboratory findings include: (1) spontaneous chromosomal breakage in peripheral T lymphocytes with rearrangements preferentially involving chromosomes 7 and 14, (2) sensitivity to ionizing radiation or radiomimetics as demonstrated in vitro by cytogenetic methods or by colony survival assay, (3) radioresistant DNA synthesis, (4) biallelic hypomorphic mutations in the NBN gene, and (5) absence of full-length nibrin protein. Microcephaly and immunodeficiency are common to DNA ligase IV deficiency (LIG4 syndrome) and severe combined immunodeficiency with microcephaly, growth retardation, and sensitivity to ionizing radiation due to NHEJ1 deficiency (NHEJ1 syndrome). In fact, NBS was most commonly confused with Fanconi anaemia and LIG4 syndrome. Genetic counselling should inform parents of an affected child of the 25% risk for further children to be affected. Prenatal molecular genetic diagnosis is possible if disease-causing mutations in both alleles of the NBN gene are known. No specific therapy is available for NBS, however, hematopoietic stem cell transplantation may be one option for some patients. Prognosis is generally poor due to the extremely high rate of malignancies.Zespół Nijmegen (Nijmegen breakage syndrome; NBS) jest rzadkim schorzeniem z wrodzoną niestabilnością chromosomową dziedziczącym się w sposób autosomalny recesywny, charakteryzującym się przede wszystkim wrodzonym małogłowiem, złożonymi niedoborami odporności i predyspozycją do rozwoju nowotworów.Choroba występuje najczęściej w populacjach słowiańskich, w których uwarunkowana jest mutacją założycielską w genie NBN (c.657_661del5). Do najważniejszych objawów zespołu zalicza się: małogłowie obecne od urodzenia i postępujące z wiekiem, charakterystyczne cechy dysmorfii twarzy, opóźnienie wzrastania, niepełnosprawność intelektualną w stopniu lekkim do umiarkowanego oraz hipogonadyzm hipogonadotropowy u dziewcząt. Na obraz choroby składają się także: niedobór odporności komórkowej i humoralnej, który jest przyczyną nawracających infekcji, znaczna predyspozycja do rozwoju nowotworów złośliwych (zwłaszcza układu chłonnego), a także zwiększona wrażliwość na promieniowanie jonizujące. Wyniki badań laboratoryjnych wykazują: (1) spontaniczną łamliwość chromosomów w limfocytach T krwi obwodowej, z preferencją do rearanżacji chromosomów 7 i 14, (2) nadwrażliwość na promieniowanie jonizujące lub radiomimetyki, co można wykazać metodami in vitro, (3) radiooporność syntezy DNA, (4) hipomorficzne mutacje na obu allelach genu NBN, oraz (5) brak w komórkach pełnej cząsteczki białka, nibryny. Małogłowie i niedobór odporności występują także w zespole niedoboru ligazy IV (LIG4) oraz w zespole niedoboru NHEJ1. Rodzice powinni otrzymać poradę genetyczną ze względu na wysokie ryzyko (25%) powtórzenia się choroby u kolejnego potomstwa. Możliwe jest zaproponowanie molekularnej diagnostyki prenatalnej jeżeli znane są obie mutacje będące przyczyną choroby. Nie ma możliwości zaproponowania specyficznej terapii, ale przeszczep szpiku może być alternatywą dla niektórych pacjentów. Generalnie prognoza nie jest pomyślna z uwagi na wysokie ryzyko rozwoju nowotworu.

An anticlastogenic function for the Polycomb Group gene Bmi1

BMI1 is a key component of multiprotein Polycomb repression complex 1 (PRC1), and its disruption in mice induces severe aplastic anemia by early adulthood. The contributing mechanisms responsible for this phenotype remain elusive. Here we show that transformed human cell lines as well as primitive hematopoietic cells exhibit a high frequency of spontaneous chromosome breaks upon BMI1 depletion and are hypersensitive to genotoxic agents. Consistent with these observations, we found that BMI1 is recruited rapidly to DNA damage foci where it blocks transcriptional elongation. We also show that BMI1 contributes to homologous recombination DNA repair and is required for checkpoint recovery. Taken together, our results suggest that BMI1 is critical for the maintenance of chromosome integrity in both normal and transformed cells.

Clinical, genetic and cytogenetic study of Fanconi anemia in an Indian population

Fanconi anemia (FA) is a rare autosomal recessive genetic disease, associated with congenital anomalies and a predisposition to cancers. FA patients exhibit spontaneous chromosome breakage and FA cells are sensitive to DNA interstrand crosslink agents and expresses high frequency of chromosome breakage. Recently 13 genes have been shown to be involved with the FA phenotype. We have carried out a detailed study in clinically diagnosed FA patients in an Indian population. Thirty three patients were clinically diagnosed with FA and had aplastic anemia and bleeding abnormalities. The genetic analysis revealed a significantly (P<0·0001) high frequency (36·4%) of parental consanguinity in FA patients compared to controls (3·33%). Chromosomal analysis revealed spontaneous chromosome breakage in 63·64% FA patients. The mitomycin C and diepoxybutane induced cultures showed a significantly (P<0·001) high frequency of chromosome breakage and radial formation compared to controls. Among 33 patients, nine (27·27%) patients developed malignancies and chromosomal abnormalities were detected in five (55·5%) patients bone marrow cells including monosomy 5 and 7, trisomy 10, der(1q) and inv(7). Cytogenetic investigation is important in aplastic anemia to rule out FA. The clinical presentation and the associated high frequency of consanguinity in FA, and the molecular analysis are complementary in the study of an Indian population.

A Role for Gcn5 in Replication-Coupled Nucleosome Assembly

Acetylation of lysine residues at the H3 N terminus is proposed to function in replication-coupled (RC) nucleosome assembly, a process critical for the inheritance of epigenetic information and maintenance of genome stability. However, the role of H3 N-terminal lysine acetylation and the corresponding lysine acetyltransferase (KAT) in RC nucleosome assembly are not known. Here we show that Gcn5, a KAT that functions in transcription, works in parallel with Rtt109, the H3 lysine 56 KAT, to promote RC nucleosome assembly. Cells lacking both Gcn5 and Rtt109 are highly sensitive to DNA damaging agents. Moreover, cells lacking GCN5 or those that express N-terminal H3 mutants are compromised for deposition of new H3 onto replicating DNA and also show reduced binding of H3 to CAF-1, a histone chaperone involved in RC nucleosome assembly. These results demonstrate that Gcn5 regulates RC nucleosome assembly, in part, by promoting H3 association with CAF-1 via H3 acetylation.

Chromosome Instability in Acute Myeloid Leukemia Is Associated to increased spontaneous breakage and Copy Number Variations (CNVs).

Abstract 4240Acute Myeloid Leukemia (AML) is a genetically pleiomorphic disease characterized by multiple genetic lesions including structural and numerical chromosome aberrations, gene mutations and changes in gene expression. The underlying mechanism behind the acquisition of these genetic abnormalities is not known, but it is likely that are facilitated by factors that increase chromosomal and genetic instability. The aim of this work was to explore chromosomal instability (CIN) in de novo AML patients by evaluating chromosome fragility and acquired DNA copy number variations (CNVs). Leukemic karyotypes were scored on bone marrow cultures (24-48 hs, without mitogens) from 24 AML patients using conventional cytogenetic and FISH. Spontaneous and FUdR -induced chromosomal fragility was studied on PHA-stimulated lymphocytes cultures (72 hs) from 10 patients and 10 healthy individuals, with and without FUdR (10mg/ml). One hundred metaphases were analyzed blind using conventional cytogenetic with sequential GTG banding. CNVs were scored on bone marrow cultures from 14 AML patients using fluorescence in situ hybridization (FISH) with two probes targeting specific regions on chromosomes 5q31 (LSI EGR/D5S721:D5S23) and 7q31 (LSI D7S522/CEP7), loci reported to be critical hotspots involved in AML. The cut-off value for FISH scoring was calculated after analyzing 250 cells from each of 10 normal bone marrow samples. The cutoff for allelic losses was equal to 0.8% for either 5q or 7q probe.Nine out of 24 AML cases presented normal karyotypes in bone marrow samples while the following abnormalities: t(3;8), t(3;11), t(4;11), t(15;17), t(9;22) and inv(16) were detected in the remaining patients. Significantly increased frequencies of spontaneous chromosome breakage, scored on untreated cultures, were detected in patients (0.22±0.03) respect to controls (0.07±0.03) (p<0.05), showing a random pattern of distribution. No differences were observed between patients and controls with FUdR treatment. Statistical analysis with Ch2 test considering data of FUdR breakpoint distribution over all individuals, identified 21 common fragile sites (c-fra) in AML cases (p<0.005). The most common sites were located at 1p32, 1p22, 1q21, 3p14, 3q27, 4q31, 5q31, 6p21 and 9q13. A high inter-individual variation in the pattern of expression was observed.Using FISH, we obtained the DNA copy numbers at 5q and 7q in all AML samples. Only losses of DNA were found with both probes. The 5q signal count in patients with normal karyotypes was on average 6.4% (range 0.5-30%), while it was 24.9% (range 0.8-90%) for patients with abnormal cytogenetic. The 7q signal count was on average 3.9% (range 0.5-10.3%) and 20.6% (range 2-63%) for cases with normal and abnormal karyotypes, respectively. Particularly, we showed that 12/14 (85.7%) AML cases, either with normal or abnormal karyotypes, exhibited copy number losses at both regions, showing different values between each patient. These findings showed that leukemic patients exhibit a CIN phenotype, providing an unstable background and facilitating the acquisition of additional genetic changes, such as CNVs, which could play an important role in disease progression. Disclosures:No relevant conflicts of interest to declare.