The identification of fanconi anemia genotypes by clastogenic stress.

Clastogen-induced chromosome damage was investigated in peripheral lymphocytes of five patients with Fanconi anemia (FA), 10 obligate heterozygotes, 25 normal controls, and four individuals with some clinical manifestations of FA. The two agents used were diepoxybutane (DEB) and mitomycin C (MMC), previously reported to be specific for the induction of increased chromosome breakage in FA cells. Following clastogenic stress, two of the five FA patients did not exhibit the expected increase in chromosomal damage while three of the four "non-FA" individuals did. In this series of subjects, the possibility of misdiagnosis is considerable when based on either clinical delineation or cytogenetic results alone. Therefore, the integration of both laboratory data and physical findings is essential before reaching a diagnosis. Furthermore, the broad range of response in both the control group and the parents of FA patients yields overlapping results, making reliable heterozygote detection impractical by these procedures.     

Effect of mitomycin C and bromodeoxyuridine on Fanconi anemia lymphocytes.

The authors studied the effect of mitomycin C (MMC) and bromodeoxyuridine (BrdU) on the induction of chromosome aberrations on lymphocytes of four patients with Fanconi anemia (FA) and of one normal subject. A control culture and six experiments were designed to test the possible synergic effect of MMC and BrdU. Their results revealed no evidence of MMC-BrdU synergism on the induction of chromosome aberrations in FA lymphocytes. However, chromosomes showed more damage when FA cells were harvested 24 h after MMC stress than when cells were harvested shortly after treatment. This can be explained by a DNA repair defect or by a toxic effect of oxygenation of cells during the procedure.     

Convergence of the fanconi anemia and ataxia telangiectasia signaling pathways

Fanconi anemia (FA) and ataxia telangiectasia (AT) are clinically distinct autosomal recessive disorders characterized by spontaneous chromosome breakage and hematological cancers. FA cells are hypersensitive to mitomycin C (MMC), while AT cells are hypersensitive to ionizing radiation (IR). Here, we identify the Fanconi anemia protein, FANCD2, as a link between the FA and ATM damage response pathways. ATM phosphorylates FANCD2 on serine 222 in vitro. This site is also phosphorylated in vivo in an ATM-dependent manner following IR. Phosphorylation of FANCD2 is required for activation of an S phase checkpoint. The ATM-dependent phosphorylation of FANCD2 on S222 and the FA pathway-dependent monoubiquitination of FANCD2 on K561 are independent posttranslational modifications regulating discrete cellular signaling pathways. Biallelic disruption of FANCD2 results in both MMC and IR hypersensitivity.     

Partial complementation of the Fanconi anemia defect upon transfection by heterologous DNA

Summary Transfectants obtained by mouse DNA-mediated gene transfer in Fanconi anemia (FA) primary fibroblasts from the genetic complementation groups A and B were examined for the frequencies of chromosomal aberrations and cytotoxicity following treatments by cross-linking agents. Cells from group A (FA 150), which is the most sensitive to such agents, are partially corrected for both the chromosomal and cellular hypersensitivity to 8-methoxypsoralen photoaddition. In contrast, after treatment with mitomycin C (MMC), only the chromosomal sensitivity is re-established to a near normal level. The opposite is true for FA group B cells (FA 145), i.e. cell survival to MMC is partially corrected, whereas the frequency of MMC-induced chromosomal aberration remains close to that of the untransfected cells. The partial phenotypic correction of the two end points examined is interpreted as indicating either a gene dosage effect or the necessity of introducing more than one gene type in order to achieve complete recovery of a normal phenotype. The phenotypic dissociation between the clastogenic and cellular hypersensitivity to crosslinking agents may offer the opportunity of isolating separately the responsible gene(s) by conventional rescue techniques.     

Differentiation of Fanconi anemia and aplastic anemia using mitomycin C test in Tunisia

Fanconi anemia (FA) is a recessive chromosomal instability syndrome that is clinically characterized by multiple symptoms. Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for FA diagnosis. In this study, we provide a detailed laboratory protocol for accurate assessment of FA diagnosis based on mitomycin C (MMC) test. Induced chromosomal breakage study was successful in 171 out of 205 aplastic anemia (AA) patients. According to the sensitivity of MMC at 50 ng/ml, 38 patients (22.22%) were diagnosed as affected and 132 patients (77.17%) as unaffected. Somatic mosaicism was suspected in an 11-year-old patient with a FA phenotype. Twenty-six siblings of FA patients were also evaluated and five of them (19.23%) were diagnosed as FA. From this study, a standard protocol for diagnosis of FA was developed. It is routinely used as a diagnostic test of FA in Tunisia.     

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.     

Evaluation of concentration and storage effects of mitomycin C in the diagnosis of Fanconi anemia among idiopatic aplastic anemia patients

BACKGROUND:

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder that shows an increased sensitivity to the intercalating agents such as mytomycin C (MMC), measured as chromosomal aberrations. This study was conducted to differentiate between FA and “idiopathic” aplastic anemia on the basis of induced chromosomal breakage study with MMC.

MATERIALS AND METHODS:

MMC stress tests in different final concentrations of 20 and 50 ng/ml of MMC were conducted on peripheral blood lymphocytes from 32 patients with aplastic anemia and 13 healthy controls. Fifty nanograms per milliliter of MMC from old, fresh and frozen stocks was used to check the sensitivity of diagnosis on FA-diagnosed patients. Statistical analysis was used for the assessment of aberrations, including chromatid and chromosome breaks and exchanges.

RESULTS:

Eight patients (25%) with a very high percentage of chromosomal breakage were diagnosed as FA on the basis of the chromosomal breakage study. Six of these patients exhibited congenital anomalies at presentation, while another two lacked such anomalies or had minor physical problems. Freshly made MMC has shown more sensitivity to detect FA patients compared with frozen or 1-week-old MMC stock.

CONCLUSIONS:

The study indicates that freshly made MMC stress test provides an unequivocal means of differentiation between FA and “idiopathic” aplastic anemia. Further, the study, the first of its kind from Iran, stresses on the need for conducting this test in all aplastic anemia cases, even those without congenital anomalies, for accurate and timely diagnosis of FA to implement appropriate therapy.     

A simple diagnostic test for Fanconi anemia by flow cytometry

A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.     

Relationship between chromosome fragility,aneuploidy and severity of the haematological disease in Fanconi anaemia

Fanconi anemia (FA) is a chromosome instability syndrome, characterized by progressive pancytopenia and cancer susceptibility. Other cellular features of FA cells are hypersensitivity to DNA cross-linking agents and accelerated telomere shortening. We have quantified overall genome chromosome fragility and euploidy as well as chromosomes 7 and 8 aneuploidy in peripheral blood lymphocytes from a group of FA patients and age-matched controls that were previously measured for telomere length. The haematology of FA samples were also characterized in terms of whole blood cell, neuthrophil and platelet counts, transfusion dependency, requirement of androgens, cortico-steroids or bone marrow transplantation, and the development of bone marrow clonal cytogenetic abnormalities, myelodysplastic syndrome or acute myeloid leukemia. As expected, a high frequency of spontaneous chromosome breaks was observed in FA patients, especially of chromatid-type. No differences in chromosomes 7 and 8 monosomy, polysomy and non-disjunction were detected between FA patients and controls. The same was true for overall genome haploidy or polyploidy. Interestingly, the spontaneous levels of chromosome fragility but not of numerical abnormalities were correlated to the severity of the haematological disease in FA. None of the variables included in the present investigation (chromosome fragility, chromosome numerical abnormalities and haematological status) were correlated to telomere length.     

Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 micrograms/ml MMC, (3)HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of (3)HdT incorporation than did those sorted from the first half of S. Fanconi's anemia cells exhibited a marked accumulation in the G(2) + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with .0.5 micrograms/ml MMC, the G(2) + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.02. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a G(2) + M increment than did FA cells, even after exposure to 0.1 micrograms/ml MMC. Examination of cells sorted from the G(2) + M peak revealed that MMC-treated FA cells were blocked prior to mitosis. To determine whether the response of FA cells was specific for bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G(2) + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.     

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.     

Specific cellular defects in patients with Fanconi anemia

Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N-methyl-N1-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.     

Impairment of homologous recombination control in a Fanconi anemia-like Chinese hamster cell mutant

DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting non-conservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.     

Cell cycle effects of the DNA topoisomerase inhibitors camptothecin and m-AMSA in lymphoblastoid cell lines from patients with Fanconi anemia

Patients with the autosomal recessive disorder Fanconi anemia (FA) present with progressive pancytopenia, skeletal abnormalities and a predisposition to leukemia. In addition to elevated rates of spontaneous chromosome aberrations occurring in cultured fibroblasts and lymphoblastoid cell lines, an increased susceptibility to DNA cross-linking agents and oxygen has been found. To explain this hypersensitivity to clastogenic agents a defective function of DNA topoisomerase I or II could be invoked, a suggestion which is supported by the co-localization of the DNA topoisomerase I gene and a putative FA gene to chromosome 20q. In order to investigate the function of DNA topoisomerases in FA, the sensitivity of lymphoid B-cell lines derived from FA patients and control cell lines to inhibitors of DNA topoisomerases I and II was compared using continuous bromodeoxyuridine labeling and bivariate Hoechst/ethidium bromide flow cytometry. Both agents inhibited cell proliferation mainly by arresting cells in the G2 phase of the cell cycle. However, no difference was found in sensitivity towards both DNA topoisomerase inhibitors between control and FA cell lines.     

范可尼贫血与泛素化

Fanconi贫血是一种罕见的隐性遗传性疾病,临床常以先天性畸形、进行性骨髓衰竭和遗传性肿瘤倾向为主要表现而确诊。FA病人细胞对DNA交联剂如丝裂霉素C (MMC)高度敏感。目前已经发现至少12种FA基因的缺失或突变能够引起FA表型的出现,其中10种相应的编码蛋白形成FA复合物共同参与FA/BRCA2 DNA损伤修复途径—FA途径。FA核心复合物蛋白FANCL具有泛素连接酶活性,在结合酶UBE2T共同作用下,催化下游蛋白FANCD2单泛化,泛素化FANCD2与BRCA2形成新的复合物,修复DNA损伤。去泛素化酶USP1在DNA修复完毕后移除FANCD2的单体泛素,使因损伤修复而阻滞的细胞周期继续进行。机体很可能在不同信号通路对FANCD2泛素化/去泛素化的精细调节下,调控FA途径参与不同的DNA修复过程。     

Cytogenetic analyses utilizing various clastogens in two sibs with Fanconi anemia,their relatives,and control individuals

Summary Structural chromosome damage, sister chromatid exchange (SCE), and proliferation kinetics were studied on lymphocyte cultures from the peripheral blood of two sibs exhibiting signs of Fanconi anemia, their relatives, and control individuals. While the rate of spontaneous chromosome breakage was at the lower limit of that known for Fanconi anemia in our patients, a distinctly greater increase than in controls of breakage frequency could be induced by isoniazid (INH), 4-nitroquinoline-1-oxide (NQO), and diepoxybutane (DEB) in their lymphocytes. Increased aberration frequencies as compared with controls were also observed in the clastogen-exposed lymphocyte cultures of the parents of both sibs, but in some experiments (NQO, DEB 24h) only in the cells of the healthy brother. There was an increase in the breakage rate of bromodeoxyuridine (BrdU)-labeled consecutive mitoses under the action of NQO, but a decrease with INH as the test clastogen.No significantly higher SCE frequency was found throughout the study in untreated and clastogen-exposed FA lymphocytes as compared with the respective controls. Proliferation was clearly inhibited by INH and NQO as indicated by a distinct increase of the percentage of BrdU-labeled first and a drastic decrease of third metaphases. The present test clastogens were shown not only to be suitable for ensuring the diagnosis of FA in patients with a low incidence of spontaneous breakage but also for determining clastogen-sensitive heterozygotes. According to these results cross-link repair cannot be the only mechanism affected by the basic defect of Fanconi anemia.Dedicated to Professor Dr. A. Barthelmess on the occasion of his 75th birthdayThis paper contains parts of the M.D. theses of D.K., H.M., and M.N.     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

Follow-up analysis of translocation and dicentric frequencies, measured by FISH-chromosome painting in breast cancer patients after partial-body radiotherapy with little bone marrow exposure

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.