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.     

Cytogenetic and molecular studies of Down syndrome individuals with leukemia.

There is an increased risk of leukemia in Down syndrome (DS) patients, with estimates ranging from 14 to 30 times the incidence rate observed for chromosomally normal children. Furthermore, one type of leukemia, called "transient leukemia" (TL), occurs almost exclusively in DS infants. The basis of the association between DS and leukemia is unknown, but we and others have hypothesized that it may be influenced by the mechanism of origin of the extra chromosome. Therefore, we initiated a cytogenetic and molecular study of nondisjunction in leukemia DS individuals. To date, we have obtained blood and/or tissue samples from 55 individuals consisting of 17 cases with TL, 7 cases of acute nonlymphocytic leukemia subtype M7 (ANLL-M7, or acute megakaryoblastic leukemia, postulated to be related to TL), and 31 cases of other forms of leukemia. Analysis of these cases suggests differences between DS children with TL and those with other types of leukemia or DS individuals with no history of leukemia. Specifically, the TL and ANLL-M7 cases have a highly significant increase in the frequency of "atypical" constitutional karyotypes (i.e., mosaic trisomies, rings, and/or isochromosomes) and are almost always male. Additionally, genetic mapping studies suggest an increase in the frequency of disomic homozygosity, especially in proximal 21q, in DS individuals with TL and ANLL-M7.     

Cytogenetic assays of chemical clastogens using mammalian cells in culture.

A protocol is described for cytogenetic assays of chemical mutagens using mammalian cells in vitro. The system employs continuous drug treatment (3 concentrations) for up to 8 h and recovery-cell populations after pulse treatments with a high dose. Both direct fixation (for recording spindle anomalies in anaphase) and colcemid-hypotonic fixation (for reading metaphase chromosome aberrations) are used in order to estimate the effects of an agent as a mitotic poison and as a clastogen respectively. Some DNA intercalating dyes (acridine orange, quinacrine mustard, neutral red) were found to be highly clastogenic whereas others (quinacrine dihydrochloride, 33258 Hoechst) are not.     

Cytogenetic,FISH and DNA studies in 11 individuals from a family with two siblings with dup(21q) Down syndrome

A Spanish family has previously been described with two siblings with dup(21q) Down syndrome. The father has a normal karyotype. The mother has a microchromosome. Cytogenetic, fluorescence in situ hybridization and DNA studies have now been carried out on the family. Findings include that the mother has three different chromosome anomalies, viz. (1) a chromosome 22 with an unusual pericentromeric region that contains alphoid DNA from chromosomes 21/13 and chromosome 22, (2) an isochromosome 21p in the frequent cell line and (3) an isochromosome 21q in a rare second cell line. A possible explanation is that the mother developed from a zygote with trisomy 21 and that mitotic error in early development resulted in the formation of two cell lines with karyotypes of 47,XX,+i(21p) and 47,XX,+i(21q), respectively. The unusual chromosome 22 represents a hitherto undescribed chromosome anomaly and one possible explanation is a translocation of the short arms between chromosomes 21/13 and 22 in the ancestry of the family. The relationship between the unusual chromosome 22 and the isochromosome formation in the mother is not known. However, all three chromosome anomalies involve the alphoid DNA of chromosome 21/13, indicating that this is not a chance finding.     

Cytogenetic studies on three pheochromocytomas derived from patients with von Hippel-Lindau syndrome

Summary Chromosomal analyses of three pheochromocytomas from patients with von Hippel-Lindau syndrome are reported. One pheochromocytoma revealed a normal karyotype, another tumor showed a trisomy 7 as the only chromosomal abnormality, whereas in a further sample a polyclonal chromosome constitution was detected. In addition to a normal 46,XX cell line, four distinct chromosomally abnormal cell lines could be identified. One cell line revealed partial trisomy for the long arm of chromosome 1 and additionally exhibited the phenomenon of telomeric association. Most interestingly, three further cell clones showed rearrangements of chromosome 3 including the region where the von Hippel-Lindau gene was mapped; three rearrangements resulted in a partial or total trisomy of 3p. Our findings are discussed in relation to previously reported cytogenetic and molecular results regarding von Hippel-Lindau syndrome.     

Cytogenetic studies in patients treated with penicillamine.

Cytogenetic studies were performed on bone-marrow cells from 11 patients with rheumatoid arthritis treated with penicillamine. One of the patients was studied while developing a granulocytopenia and thrombocytopenia. The findings show that penicillamine had no chromosome-damaging effect as estimated by the micronucleus test and by the number of structural chromosomal aberrations.     

Cytogenetic studies in fragile X syndrome]

The main purpose of this work has been to find a method which would enable the diagnosis of FXS at the cytogenetic level. The studies are based on the analysis of chromosomes from 24 cultures on RPMI-1640 base with an addition of 5-fluoro-2'-deoxyuridine (FUdR) as inhibitor of thymidylate synthetase. The results indicate, that the cultures with the addition of FUdR could considerably improve the expression of fragile X chromosome. It is of great importance, particularly un the cases in which the presence of this marker is very low. It was possibly to specify the significant percentage and the exact position of breaks, gaps and fragile sites, mostly present in autosomes. It could mean, that such factors may play a significant role, apart of X chromosome, in the pathogenesis of FXS. The results of work prove, that this kind of method could be used as a screening for cases with fragile X syndrome.     

Cytogenetic studies using Q-band polymorphisms in patients with AML receiving marrow from like-sex donors

Summary After bone marrow transplantation (BMT), it is important to monitor the bone marrow and lymphoid cell populations of the recipient to document engraftment. When donor and recipient are of unlike sex, the sex chromosomes serve as a useful marker to determine cellular origin. When donor and recipient are of like sex, autosomal heteromorphisms can be used to identify the origin of cells in metaphase. Using Q-banding, we found that 17 of 20 patient/donor pairs (85%) examined showed at least one chromosome heteromorphism that distinguished between recipient and donor cells with certainty. Five of the patients were followed up after BMT in order to document engraftment. Donor metaphases could be detected in the marrow within two weeks of BMT when the graft was successful. Chimaerism was detected in the lymphocyte population even when the graft persisted. In a case of graft failure, donor cells did not persist in the marrow, and the lymphocyte population did not convert to donor type. These studies demonstrate that autosomal heteromorphisms are useful in the study of myeloid and lymphoid chimaeric states after BMT.     

Cytogenetic studies in 12 patients with itai-itai disease.

Among 12 Itai-Itai disease patients examined, 8 patients showed a remarkably high frequency of chromatid aberrations, whereas the other 4 patients showed a much lower frequency of such aberrations, although a significant number of stable type aberrations was observed also in the latter patients. The frequencies of aneuploid cells of all 12 patients were significantly higher than those of the controls. The abnormalities were found in 50-hour and 72-hour cultures, from which it can be concluded that the aberrations occurred in the blood stem cell of the patients. In addition to these structural and numerical aberrations, satellite associations of the D and G group chromosomes were often observed.     

Cytogenetic findings in Serbian patients with Turner's syndrome stigmata

Cytogenetic findings are reported for 31 female patients with Turner's syndrome. Chromosome studies were made from lymphocyte cultures. Non-mosaicism 45,X was demonstrated in 15 of these patients, whereas only three were apparently mosaic. Eight patients showed non-mosaic and four patients showed mosaic structural aberrations of the X-chromosome. One non-mosaic case displayed a karyotype containing a small marker chromosome. Conventional cytogenetics was supplemented by fluorescence in situ hybridization (FISH) with an X-specific probe to identify the chromosomal origin of the ring and a 1q12-specific DNA probe to identify de novo balanced translocation (1;9) in one patient. To our knowledge, this is the first finding of karyotype 45,X,t(1;9)(cen;cen)/46,X,r(X),t(1;9)(cen;cen) in Turner's syndrome. The same X-specific probe was also used to identify a derivative chromosome in one patient.     

Cytogenetic studies of a family with trisomy 21 mosaicism in two successive generations as the cause of Down's syndrome

Summary A family with trisomy-21 mosaicism in two successive generations and a Down's syndrome child in the third generation is presented. Cytogenetic studies of eight individuals of this family showed a marker chromosome 15ph+ and a heteromorphic chromosome 18 in some members. The standard trisomy 21 in the proband was derived from a trisomy-21 oogonium by secondary nondisjunction in his mother.     

Cytogenetic studies in leprosy patients before and after chemotherapy

Summary The frequencies of chromosome aberrations and sister chromatid exchanges (SCEs), cell proliferation kinetics and mitotic indices were studied in peripheral blood lymphocyte cultures of leprosy patients both before and after chemotherapy. The differences in the frequencies of chromosome aberrations and SCEs between controls, paucibacillary and multibacillary patients were found to be statistically highly significant (P < 0.001). The extent of cytogenetic damage seemed to depend on the severity of the disease. Lymphocytes of untreated leprosy patients showed a low mitotic index and a slow rate of cell proliferation. Following combined treatment with dapsone and rifampicin there was an increase, but to a lesser degree (P < 0.01), in the frequency of SCEs and chromosome aberrations while the drug combination of dapsone, rifampicin and clofazamine had a non-mutagenic effect on chromosomes of the patient. Furthermore, after drug treatment, the cell proliferation rate and mitotic indices in paucibacillary patients were comparable to that of controls. These results indicate the clastogenic potency of Mycobacterium leprae and the remedial effects that follow therapeutic drug treatment.