UV-Repair is impaired in fibroblasts from patients with Fanconi's anemia

Summary Fanconi's anemia, a hereditary autosomal disease with chromosomal instability, elevated incidence of cancer and clinical symptoms is accompanied by a DNA repair deficiency. Fibroblasts from patients with Fanconi's anemia were found to be impaired in the DNA repair of UV damage. Nucleoid decondensation and recondensation after UV irradiation were less efficient in fibroblasts from patients with Fanconi's anemia than in those from a healthy proband. These data confirm our earlier findings that DNA ligase is deficient in Fanconi's anemia.     

Effects of alkylating agents on lymphocytes from controls and from patients with Fanconi's anemia

Summary The frequency of sister chromatid exchanges (SCE) and chromosome aberrations and the dynamics of cell division in peripheral blood lymphocytes of four patients with Fanconi's anemia were studied after in vitro exposure to alkylating agents TEPA and mitomycin.SCE frequency was significantly increased even after very low doses of mutagens, while chromosome aberrations were significantly increased only after high doses (0.160 g/ml mitomycin and 10^-5 M TEPA). The responses of Fanconi's anemia cells and control cells did not differ significantly. The increased frequency of both SCE and chromosome aberrations was accompanied by gradual delay of cell division, which was most conspicuous in cells from patients with Fanconi's anemia.     

Gonadal function in two siblings with Fanconi's anemia

2 siblings with Fanconi's anemia, 1 male and 1 female, aged 22 and 24 years, respectively, were evaluated at the Johns Hopkins Hospital because of short stature and hypogonadism. Plasma levels of somatomedin-C were normal in both patients, suggesting that the production of biologically active growth hormone was normal in these subjects. In addition, measurements of serum gonadotropins and plasma androgens in our patients, along with data accumulated from previous studies in the literature, show that abnormal sexual development in patients with Fanconi's anemia is due to hypergonadotropic hypogonadism.     

Position of chromosomes in the human interphase nucleus

Summary The problem of localization of chromosomes in relation to each other in the interphase nucleus of human lymphocytes was investigated by analysis of chromatid and chromosome aberrations observed in lymphocyte cultures of three patients with Fanconi's anemia, one patient with Bloom's syndrome, and in Trenimon-treated (Trenimon, Bayer) normal cells. Distribution of open gaps and breaks is highly correlated with chromosome length and distribution of breaks involved in chromatid translocations in Fanconi's anemia and in Trenimontreated cells. Both correlations are much lower in Bloom's syndrome. In Fanconi's anemia and in normal cells after Trenimon-treatment, the majority of chromatid translocations are between nonhomologous chromosomes, whereas in Bloom's syndrome mainly homologous chromosomes are involved. Statistical localization of chromosomes in relation to each other in the three-dimensional space by multidimensional scaling gives results consistent with the limited amount of independent evidence.     

Suppression of spontaneous and mitomycin C-induced chromosome aberrations in Fanconi's anemia by cell fusion with normal human fibroblasts

Summary The high rate of spontaneous and mitomycin C-induced chromosome aberrations in Fanconi's anemia fibroblasts is fully corrected after euploid somatic cell hybridization with normal human fibroblasts.     

Frequency and types of induced and spontaneous chromosome aberrations in relation to cell kinetics

Summary Induced and spontaneous structural chromosome aberrations (SCA) were studied in a child accidentally radiated with a high dose of ¹⁹²Ir, and in three sibs with Fanconi's anemia, analyzing by separate first division metaphases (FDM) and second division metaphases (SDM). The results showed that the number of SCA, number of cells with aberrations, and SCA per cell were markedly higher in FDM in all patients.Furthermore, for some type of structural changes like dicentric chromosomes and chromatid interchanges, the differences were particularly striking. The importance of ascertaining FDM identified with proper techniques, for the study of the clastogenic effect of environmental agents and some aspects related to the differences in cytogenetic features found in diverse tissues in Fanconi's anemia are discussed.     

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.     

Premature chromosome condensation in a case of Fanconi's anemia.

Evidence is presented for the occurrence of premature chromosome condensation (PCC) from micronuclei even in the first in vitro mitoses in a case of Fanconi's anemia.     

Frequency and distribution of sister-chromatid exchanges in a case of Fanconi's anemia.

In lymphocytes of a 7-year-old boy with Fanconi's anemia the frequencies and sites of sister-chromatid exchanges (SCE) were studied with the BrdU-Giemsa method. The average frequency of SCE (8.8 per metaphase) and the inter- and intrachromosomal distribution of SCE was not significantly different from the controls.     

Prevention of chromosomal breakage in Fanconi's anemia by cocultivation with normal cells

Summary The frequency of aberration in cultured lymphocytes from patients with Fanconi's anemia was significantly reduced when the cells were cocultivated with normal human lymphocytes. The results suggest that most of the chromosomal aberrations observed in cultured cells from Fanconi patients arise during cultivation and that the presence of normal cells prevents chromosomal damage by means of a hitherto unknown mechanism.     

DNA polymerases alpha beta and gamma in inherited diseases affecting DNA repair.

Fibroblasts derived from patients with diseases affecting DNA repair processes, such as Xeroderma Pigmentosum (classical and variant), Fanconi's anemia, Bloom's syndrome, Ataxia Telangiectasica, Progeria and Werner's syndrome, were assayed for the three DNA polymerases. The specific activities of these enzymes were found within the limits observed in normal human fibroblasts. Also the sedimentation properties of the three polymerases were unaltered.     

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.     

Two complementation groups of Fanconi's anemia differ in their phenotypic response to a DNA-crosslinking treatment

Summary The two genetic complementation groups reported for Fanconi's anemia (FA) correspond to two phenotypic classes as characterized by measurements of the rate of DNA semiconservative synthesis after 8-methoxypsoralen photo-addition. This test allows a rapid genetic classification of FA patients which appears to be a prerequisite for investigations of the biochemical defect(s) in FA.     

Analysis of heterogeneity in Fanconi's anemia patients of different ethnic origin

Summary Complementation studies based on mitomycin C sensitivity were performed on somatic cell hybrids between cells of a German patient with Fanconi's anemia and a Turkish, Arab, and Black African proband, respectively. Though the underlying genetic defects are expected to go back to different mutational events, the high rate of induced chromosomal aberrations in the hybrids clearly points to allelic mutations.     

Chromosomal studies of leukemic and preleukemic Fanconi's anemia patients

Summary Cytogenetic studies of three Fanconi's anemia patients are reported, one of the patients having erythroleukemia, the other two preleukemia. Clonal abnormalities were present in all three cases. Partial chromosomal duplication uncommon in other leukemias was observed. Partial duplication of the long arm of chromosome 3 has been observed in the present case of erythroleukemia examined as well as in a previously reported one.     

Antagonistic effect of cocultivation on mitomycin C-induced aberration rate in cells of a patient with Fanconi's anemia and in Chinese hamster ovary cells

Summary The rate of spontaneous chromosomal aberrations in fibroblasts of a patient with Fanconi's anemia was slightly reduced after cocultivation with Chinese hamster ovary (CHO) cells. However, after mitomycin C treatment, a significant reduction of induced chromosomal damage was found in the FA cells while a significant increase was observed in the CHO cells. This antagonistic effect could be attributed to some diffusible agent(s). The results are discussed with respect to the underlying mechanism of the disease.     

Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G2 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 G2 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 G2 prophase. Caffeine sensitivity of heterozygote lymphocytes is proposed as a new clinical test to explore heterozygosis in individuals of FA families.     

Cytogenetischer Befund und Ätiologie bei Fanconi-Anämie

Summary In addition to findings in four cases of Fanconi's anemia with hexokinase deficiency recently reported we are able to show cytogenetic results in a further case without any defect in carbohydrate metabolism.Cultures were made from peripheral blood. 30% of the mitoses showed chromatid type aberrations. Although the findings in this case no. 274 proved to be similar to those of the other patients, there are some differences, however.In this case 1. the number of chromatid breaks is smaller, 2. the number of mitoses with several coincidental aberrations is lower, 3. the number of chromatid exchanges in higher and 4. mostly the reunion is the only aberration within the metaphase (7 out of 10). It is shown that most of the cells with two chromatid breaks were able to rejoin. In numerous cells with hexokinase defect, however, several breaks without any sign of rejoining were found.This means, that in case 274 the reunion system is not damaged in the same way by an unknown genetic defect as in cells with the hexokinase deficiency. This also means, that these two types of Fanconi's anemia can be detected by different cytogenetic findings. The absence of cells with accessory nuclei in the bone marrow film is discussed in this respect, too.Our suggestion is based on results of experimental cytogenetics. Different frequencies of chromatid exchanges found in experiments on the influence of chemical mutagenes and irradiation according to the special lesion on the rejoining system are discussed.

---

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

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.