Fanconi Anemia Lymphocytes Research Articles

Improvement of genetic stability in lymphocytes from Fanconi anemia patients through the combined effect of α-lipoic acid and N-acetylcysteine

Fanconi Anemia (FA) is a rare genetic disorder, characterized by progressive bone marrow failure and increased predisposition to cancer. Despite being highly heterogeneous, all FA patients are hypersensitive to alkylating agents, in particular to 1,2:3,4-diepoxybutane (DEB), and to oxidative damage. Recent studies point to defective mitochondria in FA cells, which is closely related with increased production of reactive oxygen species (ROS) and concomitant depletion of antioxidant defenses, of which glutathione is a well-known biomarker.The objective of the present work is to evaluate the putative protective effect of α-lipoic acid (α-LA), a mitochondrial protective agent, and N-acetylcysteine (NAC), a direct antioxidant and a known precursor for glutathione synthesis, in spontaneous and DEB-induced chromosome instability (CI) in lymphocyte cultures from FA patients.For that purpose, lymphocyte cultures from 15 FA patients and 24 healthy controls were pre-treated with 20 μM α-LA, 500 μM NAC and α-LA plus NAC at the same concentrations, and some of them were exposed to DEB (0.05 μg/ml). A hundred metaphases per treatment were scored to estimate the relative frequency of spontaneous and DEB-induced chromosome breakage.The obtained results revealed that a cocktail of α-LA and NAC can drastically improve the genetic stability in FA lymphocytes in vitro, decreasing CI by 60% and 80% in cultures from FA patients and FA mosaic/chimera patients, respectively. These results suggest that the studied cocktail can be used as a prophylactic approach to delay progressive clinical symptoms in FA patients caused by CI, which can culminate in the delay of the progressive bone marrow failure and early cancer development.

Very Short Telomeres Are Characteristic of Dyskeratosis Congenita and Not Other Inherited Bone Marrow Failure Syndromes

Telomeres protect the ends of chromosomes, shorten with age, and are very short in dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) associated with mutations in telomere biology genes. “Short telomeres” were reported in Fanconi Anemia (FA), Diamond-Blackfan Anemia (DBA) and Shwachman-Diamond Syndrome (SDS) using telomere restriction fragment length or Q-FISH assays of total leukocyte or mononuclear cell DNA. These reports focused on group averages, not results from individual patients. Our objective was to determine which categories of IBMFS patients have very short telomeres, and in which leukocyte subsets, using a more sensitive and specific assay. Telomere length was measured in granulocytes, lymphocytes, naïve T-cells, memory T-cells, B-cells, and NK cells using automated multicolor flow fluorescence in situ hybridization (FISH). We previously showed that very short telomeres (<1st percentile for age) in lymphocytes, naïve T-cells, and B-cells were sensitive and specific for the diagnosis of DC (Alter et al, Blood 110:1439, 2007). Granulocytes were not specific, since in that study neutropenic patients without DC often had very short granulocyte telomeres. The current study included 53 DC patients and 87 relatives, 19 non-transplanted FA patients and 12 relatives, 21 DBA patients and 11 relatives, and 7 SDS patients and 7 relatives. There were 400 normal controls, ages 0 to 100; very short telomeres were defined as <1st percentile of normal for age. Z-scores were calculated to adjust for age; 0 is normal and -2 or below is significantly short (i.e. <2 standard deviations below the mean). Lymphocyte telomeres were <1st percentile (very short) in 96% of DC patients, 7% of DC relatives, 16% of FA patients, 5% of DBA patients, 14% of SDS patients, and none of the non-DC relatives. Granulocyte telomeres were very short in 94% of DC patients, 16% of DC relatives, 44% of FA patients, 19% of DBA patients, 14% of SDS patients, 8% of FA relatives, and none of the non-DC relatives. All three hallmark leukocyte subsets (lymphocytes, naïve T-cells, and B-cells) were very short in 81% of DC patients, 5% of DC relatives, 5% of DBA patients, and none of the FA or SDS patients or their relatives. Although 4 out of 47 (9%) IBMFS other than DC had very short telomeres in lymphocytes, only one, a patient with DBA (without mutations in known DC genes) had very short telomeres in all 3 of the hallmark lineages. Four of 87 DC relatives also had very short telomeres in all 3 lineages; they may be silent carriers in families in which DC genes have not yet been identified. The mean Z-score for lymphocyte telomeres in DC was -4.7; it was above -2 in all other categories (-0.3 in FA, -0.9 in DBA, -1 in SDS). The mean Z-score for granulocytes was -4.2 in DC, -1.9 in FA, -1.2 in DBA, and -1.5 in SDS. Thus, based on the Z-scores, the average telomere length was very short in DC lymphocytes and granulocytes; normal in FA lymphocytes and borderline in FA granulocytes; and normal in DBA and SDS lymphocytes and granulocytes. Flow-FISH provides analysis of multiple cell types in individual patients, and identification of specific individuals with very short telomeres, as well as the age-adjusted mean telomere length in a diagnostic subset. No FA or SDS patients, and only one with DBA, met the DC diagnostic criteria of very short telomeres in three or more lymphocyte subsets. The telomere length deficit in DC appears to be more severe and more frequent than in the other inherited bone marrow failure syndromes.

Fanconi anemia lymphocytes: effect of dl-α-tocopherol (Vitamin E) on chromatid breaks and on G2 repair efficiency

The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells.The effect of 100μM dl-α-tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample.α-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%).The efficiency of the G2 repair process (G2R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2R rate, both in FA cells and controls.These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells.

Frequencies of X-ray induced chromosome aberrations in lymphocytes of xeroderma pigmentosum and Fanconi anemia patients estimated by Giemsa and fluorescence in situ hybridization staining techniques

Blood lymphocytes from xeroderma pigmentosum (XP) and Fanconi anemia (FA) patients were assessed for their sensitivity to ionizing radiation by estimating the frequency of X-ray (1 and 2 Gy)-induced chromosome aberrations (CA). The frequencies of aberrations in the whole genome were estimated in Giemsa-stained preparations of lymphocytes irradiated at G0 or G2 stages. The frequencies of translocations and dicentrics involving chromosomes 1 and 3 as well as the X-chromosome were determined in slides stained by fluorescence in situ hybridization (FISH) technique. An increase in all types of CA was observed in XP and FA lymphocytes irradiated at G0 when compared to controls. The frequency of dicentrics and rings was 6 to 27% higher (at 1 and 2 Gy) in XP lymphocytes and 37% higher (at 2 Gy) in FA lymphocytes than in controls, while chromosome deletions were higher in irradiated (30% in 1 Gy and 72% in 2 Gy) than in control XP lymphocytes and 28 to 102% higher in FA lymphocytes. In G2-irradiated lymphocytes the frequency of CA was 24 to 55% higher in XP lymphocytes than in controls. In most cases the translocation frequencies were higher than the frequencies of dicentrics (21/19).

Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients

Fanconi anemia (FA) is characterized at the cellular level by a high frequency of spontaneous chromosomal aberrations; crosslinking agents cause an abnormal increase in the frequency of chromosomal damage, and semiconservative DNA synthesis is severely inhibited. Deoxyribonucleotides are needed in both semiconservative and repair DNA synthesis. To investigate the involvement of deoxyribonucleotide pools in the inhibition of DNA synthesis in FA, we evaluated the effect on FA lymphocytes of hydroxyurea (HU), an inhibitor of ribonucleotide reductase which is known to alter the intracellular levels of deoxyribonucleotides. To achieve this goal, lymphocyte cultures of 4 FA patients and 4 normal individuals were used. Cultures were treated with HU and/or mitomycin C and normal human plasma. All cultures were processed to detect the number of DNA synthesizing nuclei by autoradiography. Scoring of 2000 nuclei for each kind of culture every 6 h in the last 24 h of incubation showed that, in long incubation periods, DNA synthesis in FA is largely inhibited by HU and this hypersensitivity may be partially decreased by addition of normal human plasma. It is known that recovery from damage induced by HU involves several enzymes such as flavin oxido-reductase, superoxide dismutase and catalase which are involved in the production or scavenging of O 2 radicals; FA cells are deficient in the detoxification of oxygen and this could explain the response of FA cells to HU.

Fragile sites and chromosome instability: the distribution of breaks induced by cis-diamine-clichloro-platinum (II) in Fanconi anemia lymphocyte cultures

The distribution of chromosome breaks induced by the antitumor drug cis-diamine-dichloroplatinum (II) in lymphocyte cultures from Fanconi anemia patients was analyzed. Breakpoints occurred nonrandomly over an arbitrarily defined human karyotype of 319 bands. These bands were classified according to either their banding pattern or their fragile site status (whether or not a fragile site of a given type is located at a chromosomal band). A significant involvement of G-light and fragile site bands was detected. The preferential occurrence of breaks at fragile site bands was limited to common fragile site bands (essentially of the aphidicolin-type).

Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G 2 prophase

In this investigation peripheral blood lymphocytes from 3 Fanconi's anemia (FA) patients, 2 FA heterozygotes and 4 normal subjects were treated with caffeine and/or adenosine, and/or niacinamide during G 2 prophase. Caffeine dramatically increased breakage levels in homozygote and heterozygote cells. Niacinamide and adenosine decreased the amount of chromosomal aberrations detected in FA homozygote and heterozygote lymphocytes treated and untreated with caffeine during G 2 prophase. Caffeine sensitivity of heterozygote lymphocytes is proposed as a new clinical test to explore heterozygosis in individuals of FA families.

Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G 2 prophase

In this investigation peripheral blood lymphocytes from 3 Fanconi's anemia (FA) patients, 2 FA heterozygotes and 4 normal subjects were treated with caffeine and/or adenosine, and/or niacinamide during G 2 prophase. Caffeine dramatically increased breakage levels in homozygote and heterozygote cells. Niacinamide and adenosine decreased the amount of chromosomal aberrations detected in FA homozygote and heterozygote lymphocytes treated and untreated with caffeine during G 2 prophase. Caffeine sensitivity of heterozygote lymphocytes is proposed as a new clinical test to explore heterozygosis in individuals of FA families.

Cytogenetic toxicity of paraquat and streptonigrin in Fanconi's anemia.

We reported earlier that the frequency of chromosomal aberrations observed in Fanconi's anemia lymphocyte cultures depends on the oxygen tension during growth of the cultures, suggesting that "activated oxygen species" (superoxide, O2-; hydrogen peroxide, H2O2; hydoxyl radical, OH; and singlet oxygen, 1O2) or other reactive products generated during oxygen metabolism may be involved in the production of chromosomal damage in this syndrome. Paraquat and streptonigrin, agents that have been proposed as model compounds exerting cellular toxicity through overproduction of superoxide, were tested for their clastogenic potency in lymphocyte cultures from healthy controls and patients with Fanconi's anemia. Paraquat, at concentrations that severely affected mitotic activity (100-200 micrograms/ml), appeared to be a weak clastogen in human lymphocytes, whereas a clastogenic effect of streptonigrin was demonstrable already at a concentration as low as 0.005 microgram/ml. The results indicate that Fanconi's anemia lymphocytes fail to exhibit an increased sensitivity to the antimitotic and clastogenic effects of paraquat and streptonigrin. This suggests that intracellular superoxide is not critically involved in the generation of spontaneous chromosomal aberrations in Fanconi's anemia. We infer from these and previous data that singlet oxygen (1O2) may be a critical contributor to the chromosomal breakage in this disorder.

Clastogenicity of cyclophosphamide in Fanconi's anemia lymphocytes without exogenous metabolic activation

The indirect mutagen cyclosphosphamide (CP) is known to induce chromosomal aberrations in human lymphocyte cultures only after exogenous metabolic activation. We document here that lymphocytes from five patients with Fanconi's anemia (FA) exhibited extensive chromosomal breakage when cultured in the continuous presence of 1 mM CP without deliberate activation of the drug, whereas, cultures from healthy subjects were not significantly susceptible to breakage under these conditions. CP exposure also enhanced the frequency of sister chromatid exchanges (SCE), but to similar extents in control and FA subjects, suggesting that there is a similar low level of CP metabolism in FA and normal lymphocytes. The direct clastogenic effect of nonactivated CP as demonstrated here in FA lymphocyte cultures may be of value in screening for possible hypersensitivity in patients considered for CP therapy.

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.

Effect of oxidants and antioxidants on chromosomal breakage in Fanconi anemia lymphocytes.

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.

Proliferative kinetics and mitomycin C-induced chromosome damage in Fanconi's anemia lymphocytes.

Lymphocytes from two sisters with Fanconi's anemia (FA) were studied for cell cycle kinetics, sister chromatid exchanges (SCEs), and chromosomal aberrations when they had undergone one, two, or three or more divisions in mitomycin C (MMC)-treated cultures. Lymphocytes from the parents, another sister of the probands, and a healthy unrelated adult were examined as controls. Analyses of cell cycle kinetics by the sister chromatid differential staining method revealed that the relative frequency of metaphase cells at their third or subsequent divisions was much smaller in untreated FA cultures than in normal cultures fixed at 96 h after phytohemagglutinin stimulation. These data indicate that FA cells proliferate much more slowly than normal cells. MMC treatments of FA and normal cells led to a clearly dose-related delay in cell turnover times, the duration of delay being much longer in FA than in normal cells. FA cells had about 1.4 times higher frequencies of SCEs than normal cells in both MMC-treated and untreated cultures. FA cells also showed several times higher frequencies of chromosomal aberrations than normal cells, and the frequency of chromosomal aberrations decreased through subsequent mitoses by approximately 60% in both FA and normal cells.

First Announcement of the Fanconi Anemia International Registry

We, Arleen D. Auerbach, USA, and Traute M. Schroeder, West Germany, call upon our colleagues to cooperate with us in order to establish and maintain an International Registry of all known Fanconi anemia (FA) patients. Clinical diagnosis of FA is often difficult to establish due to phenotypic diversity of patients and the occurrence of other disorders that manifest some of the features of FA. Diagnosis may be confirmed by the use of a cytogenetic method based on the sensitivity of FA lymphocytes, fibroblasts, and amniotic fluid cells to chemically induced chromosome breakage (Auerbach, Adler, and Chaganti: Pediatrics 67:128, 1981).

First Announcement of the Fanconi Anemia International Registry

We, Arleen D. Auerbach (USA) and Traute M. Schroeder (West Germany), call upon our colleagues to cooperate with us in order to establish and maintain an International Registry of all known Fanconi anemia (FA) patients. Clinical diagnosis of FA is often difficult to establish because of the phenotypic diversity of patients and the occurrence of other disorders which manifest some of the features of FA. Diagnosis may be confirmed by the use of a cytogenetic method based on the sensitivity of FA lymphocytes, fibroblasts, and amniotic fluid cells to chemically induced chromosome breakage (Auerbach et al., Pediatrics 67: 128, 1981). The aim of the registry is to ascertain, as far as possible, all presumptive FA patients and to gather genetic, hematologic, and clinical data in order to eventually define the heterogeneity of the disorder, establish a means of exact differential diagnosis, and study the clinical course of the disease, including the incidence of malignancy.

First announcement of the Fanconi anemia International Registry

We, Arleen D. Auerbach (USA) and Traute M. Schroeder (West Germany), call upon our colleagues to cooperate with us in order to establish and maintain an International Registry of all known Fanconi anemia (FA) patients. Clinical diagnosis of FA is often difficult to establish because of the phenotypic diversity of patients and the occurrence of other disorders which manifest some of the features of FA. Diagnosis may be confirmed by the use of a cytogenetic method based on the sensitivity of FA lymphocytes, fibroblasts, and amniotic fluid cells to chemically induced chromosome breakage (Auerbach et al., Pediatrics 67: 128, 1981). The aim of the registry is to ascertain, as far as possible, all presumptive FA patients and to gather genetic, hematologic, and clinical data in order to eventually define the heterogeneity of the disorder, establish a means of exact differential diagnosis, and study the clinical course of the disease, including the incidence of malignancy.

Mitomycin C Test for Diagnostic Differentiation of Idiopathic Aplastic Anemia and Fanconi Anemia

A cytogenetic test was developed to enable the diagnostic differentiation between Fanconi anemia and the group of disorders classified as "idiopathic" aplastic anemia. The method is based on the effect of the bifunctional alkylating agent mitomycin C (MMC) on chromosomes of peripheral lymphocytes in culture 92 to 96 hours. At the highest concentration of 80 ng/ml of MMC, about a 50-fold increase of chromosomal breaks and a 200-fold increase of radial figures was observed in Fanconi anemia lymphocytes in comparison with the average values for aplastic anemia cells or cells of healthy control subjects. In addition, sister chromatoid exchange analysis of cells treated with MMC demonstrated a marked inhibition of mitotic activity of lymphocytes of patients with increased sensitivity to MMC. It is advocated that the MMC-stress test should be used in each case of pancytopenia, as part of the therapy planning. The cytoreduction regimens used prior to bone marrow transplantation may produce lethal complications in patients with high clastogenic response to MMC.

Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 mug/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges in accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage.