Spontaneous and induced chromosomal instability in Werner syndrome

Summary In extension of a previous study, spontaneous and clastogen-induced chromosome damage was analyzed in cultures of peripheral blood lymphocytes from six further patients with Werner syndrome (WS) and six healthy controls. In addition, sister chromatid exchange (SCE) was estimated in four of these cases. Lymphocytes of patients with various other diseases were used for another series of control experiments. Diepoxybutane (DEB), 4-nitroquinoline-1-oxide (NQO), and bleomycin (BLM) were the standard clastogens throughout the study. While the spontaneous frequency of chromosomal breakage was significantly higher in lymphocytes from all the patients than in the control cells, the basis SCE rate was un-affected in WS cells. Sensitivity of WS cells to the chromosome-damaging action of BLM did not differ from that of control cells, and their sensitivity to DEB was slightly greater than that of control lymphocytes. However, NQO induced a more distinct increase of both break and interchange aberrations in the WS cells than in control cells or cells from patients with other diseases. This effect was not found for the SCE rate. Our data demonstrate the exceptional cytogenetic features of this syndrome: Although the spontaneous and the DEB- and NQO-induced chromosomal breakage rate would suggest that WS is like a classic chromosomal instability syndromes, the lack of sensitivity of WS cells to bleomycin and their stable SCE frequency compared with that of control cells clearly delimitate this syndrome from other entities.     

Preliminary communication: prenatal detection of the Fanconi Anemia gene by cytogenetic methods.

We have studied the pattern of chromosome instability in cultured fibroblasts and fetal membrane cells from a fetus aborted by an individual with a history of a previous child affected with Fanconi anemia (FA). These cells exhibited a low level of spontaneous chromosome instability. Upon treatment with diepoxybutane (DEB), chromosome breakage increased to a level comparable to that reported earlier in DEB-treated FA heterozygous cells. Cultured cells derived from chromosomally normal fetuses which served as controls did not show DEB-induced chromosome breakage. This observation suggests that the fetus studied is heterozygous for the FA gene. The ability to distinguish readily between the three genotypes (homozygous FA, heterozygous FA, and normal) in an in vitro stress system that measures the response of the cells to a clastogenic agent makes available a test for the prenatal and postnatal detection of the FA gene.     

Normal red blood cells partially decrease diepoxybutane-induced chromosome breakage in cultured lymphocytes from Fanconi anaemia patients

Objectives: Fanconi anaemia (FA) is a cancer‐prone chromosome instability syndrome characterized by hypersensitivity to DNA cross‐linking agents, such as diepoxybutane (DEB). Previous studies have shown that normal red blood cells (RBC) can protect cultured lymphocytes against chromosomal breaks induced by DEB. The present study was designed to analyse influence of RBCs from normal individuals on frequency of DEB‐induced chromosome breaks in lymphocyte cultures from FA patients. Materials and methods: A comparative study was performed between DEB‐induced chromosome breaks in cultures of FA lymphocytes with either autologous or heterologous RBCs. A further comparative study was carried out between whole blood cultures from FA patients performed on two occasions, before and 1 week after transfusion of RBCs. Results: It was observed that normal RBCs compared to FA RBCs, partially reduced chromosome breaks in cultured FA lymphocytes. A significant reduction in DEB‐induced breaks was also observed in FA cultured lymphocytes obtained 1 week after transfusion of RBCs, in comparison to those observed in the same patients before RBC transfusion. Conclusions: This study shows that DEB‐induced chromosome instability in FA lymphocytes is partially reduced by normal RBCs. This effect may have some clinical relevance in vivo, whenever FA patients receive a RBC transfusion.     

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 oxidants and antioxidants on chromosomal breakage in Fanconi anemia lymphocytes

Summary Peripheral blood lymphocytes from eight Fanconi anemia (FA) patients, 14 FA heterozygotes, and nine normal subjects have been tested for their susceptibility to chromosomal breakage induction by diepoxybutane (DEB) and by two peroxides. In addition, the effect of five antioxidants was investigated in standard cultures and in cultures stressed either with DEB or with butylhydroperoxide (BHP) or with hydrogen peroxide (H₂O₂). DEB, BHP, and H₂O₂ dramatically increased the chromosomal breakage levels in homozygous and heterozygous FA cells. A partial correction of chromosomal instability was obtained by treating the patients' lymphocytes with antioxidants. A protective effect was also noted in the DEB or peroxide-stressed lymphocytes of patients and heterozygotes, grown in the presence of antioxidants.     

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.     

The effect of aphidicolin on Fanconi's anemia lymphocyte chromosomes

The cytogenetic effect of the DNA polymerase alpha inhibitor aphidicolin (APC) at a dose which did not affect cell cycle progression was determined in normal and Fanconi's anemia (FA) lymphocytes. APC enhanced sister-chromatid exchange (SCE) levels by about twice both in control and FA cells, while the yields of chromosome breakage increased up to 20 times in normal lymphocytes and 4 times in FA cells. APC did not act synergistically with the bifunctional alkylating diepoxybutane in terms of SCE either in normal or in FA lymphocytes. However, chromosome aberrations in cultures from normal subjects were much more than expected by an additive mode of action.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

Cytogenetic sensitivity of G0 lymphocytes of Fanconi anemia patients and obligate carriers to mitomycin C and ionizing radiation

It is well known that Fanconi anemia (FA) patients show a hypersensitivity to the effect of cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), while the sensitivity of these patients to ionizing radiation is still controversial. Fanconi anemia heterozygotes do not show a hypersensitivity to the above mentioned agents compared to normal individuals. To examine the radio-sensitivity of Fanconi anemia patients and heterozygotes, ten patients and 13 heterozygotes were enrolled in this study. Standard metaphase analysis for detection and verification of radio-sensitivity was used to establish the relationship between gamma-ray and chromosome breakages in these groups. Statistical analysis was used for the assessment of aberrations including chromatid and chromosome breaks and exchanges. Results of chromosome aberration yield that: (i) differentiation between obligate carriers and the control group after MMC treatment and gamma irradiation was not possible; (ii) homozygotes were clearly distinguishable from heterozygotes and controls after MMC treatment; (iii) FA patients don't show hypersensitivity to gamma irradiation compared to normal controls and heterozygous carriers.     

Partial correction of chromosome instability in Fanconi anemia by desferrioxamine

Summary The action of the iron chelator desferrioxamine (DFO) on the cytogenetic pattern of cultured lymphocytes from Fanconi anemia (FA) patients was investigated. The addition of 10^-4 M DFO throughout the culture time resulted in a 50% reduction of the spontaneous chromosome breakage of FA cells. In addition, the clastogenic action of diepoxybutane on FA lymphocytes was also partly counteracted by DFO. The above findings support the assumption that one of the mechanisms involved in the pathogenesis of FA might be an impaired capacity of the cells from such patients to remove active oxygen species. The relationship between intraleukocyte chelatable iron pool and free radical formation in FA subjects is discussed.     

Role of haemoglobin in the protection of cultured lymphocytes against diepoxybutane (DEB), assessed by in vitro induced chromosome breakage

Diepoxybutane (DEB) is an alkylating agent that can be used to assess chromosome instability in repair-deficient subjects. Previous authors investigated the role of red blood cells (RBC) in determining individual susceptibility to DEB in normal healthy donors, and demonstrated that a polymorphic enzyme in RBC, Glutathione S-transferase T1 (GSTT1), is involved in DEB detoxification. In the present work we studied the influence of individual GSTM1 and GSTT1 genotypes and the presence of RBC on the frequency of DEB-induced chromosome breakage in lymphocyte cultures from normal individuals and, in particular, the influence of isolated components of RBC: RBC membranes, RBC lysate, and haemoglobin. Our results confirm that individual GSTT1 genotypes modulate the level of genetic lesions induced by DEB; however, this effect was not sufficient to explain the highly significant variation in chromosome breakage between whole blood and RBC-depleted cultures. We showed that RBC can protect cultured lymphocytes against chromosome breakage induced by DEB and we demonstrated the particular role of haemoglobin in the protective effect.     

Spontaneous 6-thioguanine-resistant lymphocytes in Fanconi anemia patients and their heterozygous parents

Summary The incidence of spontaneous 6-thioguanine-resistant (TG^r) lymphocytes was studied in the peripheral blood collected from seven Fanconi anemia (FA) patients and five of their heterozygous parents using an autoradiographic or a lymphocyte cloning method. Five of the seven patients showed a significantly elevated incidence of TG^r lymphocytes as compared to age- and sex-matched healthy controls. There was, however, no difference between FA heterozygotes and controls. These results suggest some variability among the patients similar to those reported in clinical and cytogenetic investigations. The basis for the increase in TG^r cells in the patients is not known, but the inherent genomic instability reflected as increased frequencies of chromosomal aberrations is one possible explanation.     

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.     

The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced chromosome breaks

Diepoxybutane (DEB) is an established mutagen that induces chromosome damage following in vitro treatment of peripheral blood lymphocytes. It is widely used to identify patients with Fanconi Anemia (FA), a clinical situation that is characterized, besides the hypersensitivity to DEB, by an elevated foetal haemoglobin (HbF) content in the peripheral blood. In a previous study, we showed that red blood cells (RBC) from normal individuals can protect cultured lymphocytes against chromosomal breaks induced by DEB and demonstrated the particular role of haemoglobin in the protective effect. In the present work, we studied the influence of RBC extracted from umbilical cord blood of neonates (F cells) on the frequency of DEB-induced chromosome breaks in lymphocyte cultures from normal individuals. Simultaneously, we determined individual GSTT1 and GSTM1 genotypes and the activity of Pi-class glutathione S-transferase (GSTP), catalase and superoxide dismutase (SOD) in adult and foetal RBC. Our results show that F cells, in comparison with adult RBC, elicit a better protection of cultured lymphocytes from normal individuals against chromosome breaks induced by DEB. Variability in the protective effect among RBC from different individuals was observed; we confirmed that the GSTT1 genotype modulates this inter-individual variability, but it is not sufficient to explain all of the protective effect of F cells. Our results suggest that the increased protective effect of F cells can be, at least in part, correlated with an increase in the activity of glutathione S-transferase, catalase and superoxide dismutase, in particular Cu/Zn SOD, in F cells compared with adult RBC.     

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.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.     

Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells.

BrdU-Hoechst flow cytometry was employed to study the proliferation kinetics of blood lymphocytes from patients with Fanconi anemia (FA). Compared to controls, untreated FA lymphocytes show normal response to PHA stimulation, normal G0/G1 exit rates, and normal first S-phase durations. The G2 phase of the first cell cycle, however, is severely prolonged, and 24% of the recruited population become arrested during the first chromosome cycle (S, G2/M phases). The delay suffered during G2 appears to be compensated in part by a subsequent G1 phase duration that is unusually short for postnatal human cells (3.7 +/- 0.5 hrs). In analogy to what has been observed in other cell systems after experimental delays of the chromosome cycle, we therefore postulate that at least some FA cells enter their second growth phase without prior completion of the delayed chromosome cycle. Renewed replication would ensue in such cells without prior passing through mitosis and cytokinesis, leading to endoreduplication, which is a frequent finding in the FA syndrome.     

Mutagenic effects of isonicotinic acid hydracide in Fanconi's anemia

Summary Isonicotinic acid hydracide (INH) increases substantially the chromosomal instability in Fanconi's anemia (FA) cells. The same concentrations of INH do not significantly break chromosomes in heterozygous or normal cells. INH does not induce alkylation or cross-links in the DNA like other mutagens known to increase breakage in FA cells. Possible mechanisms of the effect of INH are discussed. One consequence of this experiment is the possibility of an exact and doubtless prenatal diagnosis of a homozygous FA fetus.     

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.