Spontaneous expression of fra(10)(q25) in bone marrow from a patient with agranulocytosis

We report on the spontaneous expression of fra(10)(q25) in bone marrow from a patient with agranulocytosis. Expression of this fragile site in both bone marrow and leukocytes was enhanced by bromodeoxyuridine (BrdU), while folic-acid-deficient medium enhanced the expression of fra(10)(q25) only in leukocytes. Variability in the expression of fra(10)(q25) in bone marrow and leukocyte cultures over an 18-month period was also found.     

Differential expression of fragile site Xq27 in cultured fibroblasts from hemizygotes and heterozygotes and its implications for prenatal diagnosis

Expression of the fragile site Xq27 (fraXq27) was studied in metaphases derived from fibroblasts of 8 hemizygotes and 2 heterozygotes, cultured in 2 different media containing reduced concentrations of folic acid. The proportion of fra(X) (q27)-positive metaphases ranged from 1.0 to 14%, which is considerably lower than in lymphocyte cultures from the same individual, but differed with respect to the culture medium used. Four hemizygotes showed a higher proportion of fra(X)-positive cells in medium 199 than in methotrexate-exposed cultures. No fra(X)-negative cultures were observed when the folic acid inhibitor methotrexate was added to medium 199. Thus, in all 10 individuals carrying the fra(X), the fragile site could be detected in fibroblast cultures. We conclude that the expression of fra(X) (q27) in cultured fibroblasts should be studied using at least 2 types of culture conditions, because familial, i.e. genetic differences may influence the expression of this fragile site in fibroblasts. The implications for prenatal diagnosis are discussed on the basis of investigations in 15 pregnancies at risk for the trait.     

Bromodeoxyuridine induces chromosomal fragile sited in the canine genome

Peripheral blood lymphocytes from 3 clinically normal domestic dogs were cultured for bromodeoxyuridine (BrdU) induction of fragile site expression. BrdU induced fragile site expression in cells from all 3 dogs. The mean percent of cells with fragile sites and the mean number of fragile sites per cel were significantly increaed in all BrdU incubated cultures compared to control cultures. The frequency of BrdU fragile site expression did not vary significantly among the dogs. Lymphocytes from all 3 dogs expressed BrdU induced autosomal fragile sites. Two BrdU induced fragile sites were identified on the long arm of chromosome 1, one of which was close to or coincident with a previously identified folate sensitive fragile site on this canine chromosome. Lymphocytes from the 2 female dogs also expressed BrdU induced fragile sites on the X chromosome, but BrdU failed to induce fragile sites on the X chromosome from the one male dog in the study. The 2 BrdU-induced fragile sites identified on the long arm of the X chromosome were close to, or coincident with 2 previously described folate-sensitive common fragile sited on the canine X chromosome. This is the fist report of induction of BrdU-inducible fragile sites in the genome of the domestic dog. © 1993 Wiley-Liss, Inc.     

Common fragile sites: their prevalence in subjects with constitutional and acquired chromosomal instability

Chromosomal fragile sites that are inducible by methotrexate and aphidicolin are frequent in the human population. To assess the frequency and distribution of these common fragile sites, we performed a cytogenetic survey on lymphocytes from subjects known to be particularly prone to breakage because of constitutional chromosomal instability, the possession of a rare fragile site, or Fanconi anemia. Furthermore, a group of cancer patients was included in this study in view of possible acquired chromosomal instability. Lymphocyte chromosomes from several healthy donors were analyzed under identical conditions. We found that methotrexate- and aphidicolin-induced fragile sites are widespread in the general population, showing a similar breakpoint distribution. Ten fragile sites (3p14, 16q23, 2q32, 6q25, 4p16, 4q31, 14q24, 1p31, 20p12, 7q21) were observed in at least 40% of the individuals among the different groups. Our data point out a significantly increased breakage induced by aphidicolin in lymphocytes from cancer patients and, to a lesser extent, from rare fragile sites carriers. These results suggest that common fragile sites are enhanced in some constitutional and acquired conditions.     

Constitutive fragile sites in fra(X) individuals

Recently, it was proposed that the constitutive fragile site at 3p14 be used as an "internal control" to indicate the effectiveness of the FUdR fragile site induction system. We have tested this hypothesis by determining the frequency of constitutive fragile sites at 1p31, 3p14, and 16q23 in cultures from 42 known fra(X) individuals. At least 50 cells were analyzed from each case. Seventy-four percent (31/42), 95% (40/42) and 90% (38/42) of the fra(X) individuals exhibited frequencies of less than 4% at constitutive fragile sites 3p14, 1p31 and 16q23, respectively. Of the 42 individuals tested, 12 or 28.6% showed no fragility at any of the 3 sites studied. On the other hand, at least one constitutive fragile site was observed in 50 cells studied from over 70% of the 42 people studied. It is suggested that "positive controls" continue to be used, while at the same time recording all fragile sites to identify a combination of constitutive fragile sites that may serve as an internal control indicator, and that DNA marker studies be used to complement cytogenetic testing.     

Heritable fragility at 11q13 and 12q13

The chromosomes of two mentally retarded probands were investigated because they were suspected of having the fragile X syndrome. However two other fragilities were detected. In one patient a fra(11)(q13) was found and in the other a fra(12)(q13). Family studies revealed that both fragile sites were real heritable ones. Besides these two heritable fragile sites, the common fragile site at 3p14 was frequently observed. The effects of BUdR, FUdR and methotrexate on the frequency of the three fragilities were studied. The two heritable fragile sites differed from the common fragile site at 3p14 with respect to their inducibility by FUdR and methotrexate.     

A new family with fra(10)(q25): Spontaneous expression and 100% expression with 100 μM BrdU

We report on a family with fra(10)(q25) ascertained through a female with multiple minor anomalies and present in her phenotypically normal father and other family members. The site is expressed spontaneously at levels of 9% in lymphocytes, 2% in lymphoblasts, and 6% in skin fibroblasts. Expression frequency is significantly enhanced to levels as high as 100% by addition of BrdU to the culture medium. The consistently high level of induced expression in lymphoblasts from the proband facilitates analysis of the biochemical and structural bases of fragile site expression.     

A new family with fra(10)(q25): spontaneous expression and 100% expression with 100 microM BrdU

We report on a family with fra(10)(q25) ascertained through a female with multiple minor anomalies and present in her phenotypically normal father and other family members. The site is expressed spontaneously at levels of 9% in lymphocytes, 2% in lymphoblasts, and 6% in skin fibroblasts. Expression frequency is significantly enhanced to levels as high as 100% by addition of BrdU to the culture medium. The consistently high level of induced expression in lymphoblasts from the proband facilitates analysis of the biochemical and structural bases of fragile site expression.     

Heritable fragile sites and cancer: fra(16)(q22) in lymphocytes of an acute nonlymphocytic leukemia patient with inv(16)(p13q22)

Fragile site testing was performed on normal peripheral blood lymphocytes from three acute nonlymphocytic leukemia patients who carried inv(16)(p13q22) in malignant cells. Cultures were treated with BrdU, distamycin A, Hoechst 33258, or folic acid deprivation to induce fragile site expression. One patient was found to be a carrier of fra(16)(q22), but the expression was observed only by Hoechst 33258 treatment.     

Fragile site expression in families with von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that predisposes to diverse tumors including renal cell carcinoma. Six affected and four unaffected subjects from five families were studied to determine the frequency of fragile site expression. Peripheral lymphocyte cultures from each subject were treated with low folate, 5-fluorodeoxyuridine (FUdR), and FUdR plus caffeine for fragile site induction. A site was considered to be fragile if it was expressed at least two times in half of the affected or unaffected subjects. Of the established sites, four were expressed in the unaffected group (3p14, 6p22, 8q22, and Xp22) and six were expressed in the affected group (3p14, 4q31, 5q31, 7q32, Xp22, and Xq22). Only 3p14 and Xp22 were expressed in both groups. There were four new sites: three (3q26, 6p21, 7p15) in the unaffected group and one (16q24) in the affected group. The 3p14 site was expressed twice as frequently in affected versus unaffected subjects. This finding is of interest because of reports of the involvement of 3p14 in hereditary renal cell carcinoma and in VHL.     

Common fragile sites in chromosomes of bone marrow cells and peripheral blood lymphocytes from healthy persons and leukemia patients

Frequency and distribution of aphidicolin-induced fragile sites (c-fra) on chromosomes of both peripheral blood lymphocytes (PBL) and bone marrow (BM) from 15 leukemia patients were studied in comparison with 22 PBL and six BM samples from healthy volunteers. In normal controls, the most frequent c-fra was 3p14 in PBL, but it was 4q21-25 in BM. The second most frequent site was 16q23 in PBL, but it was 7q11.2 in BM. These differences in fragile sites between PBL and BM may be related to distinct functions of cells in different tissues. The total number of breaks in PBL and BM showed a significant difference among individuals, but the sites were generally common. The frequency of breaks in PBL from leukemia patients was higher than in controls when the leukemic cells had any karyotypic abnormalities. In leukemia without karyotypic abnormalities and acute myeloid leukemia (AML) with (15:17), the frequency of breaks fell within normal or slightly above normal ranges. Breaks at 3p14 (22.0% of total breaks), 16q23 (7.3%), 7q32 (4.3%), Xp22 (3.7%), and 6q26 (2.9%) were frequent in PBL from seven AML patients. Breaks at 4q21-25 (2.1%), 7q22 (2.2%), 7q32, and Xp22 were more frequently induced than in controls, and 1p32 (0.1%), 3p14, 6q26, and 16q23 were less often expressed than in controls. On the other hand, PBL from acute lymphoblastic leukemia patients showed a higher frequency of breaks only at 1p22 (3.4%) and the frequency of breaks at 3p14 (30.2%) decreased (p less than 0.05). The PBL from AML patients with t(8;21) (q22;q22) showed breaks at 8q22 and 8q24, and the frequencies were significantly higher than those of other types of leukemia or in controls (p less than 0.001). The results of this study suggest that fragility of chromosomes may be related to the chromosomal rearrangement in or predisposition to leukemia.     

Investigation of genetic susceptibility to non-small cell lung cancer by fragile site expression

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.     

Analysis of chromosome breakage in the Prader-Labhart-Willi syndrome

Analysis of chromosome breakage with mitomycin C (MMC) and folate-deficient culture conditions was undertaken on 18 Prader-Labhart-Willi syndrome (PLWS) patients (10 with 15q12 deletion [5 females, 5 males; mean age = 17.9 yr, range of 0.3 to 40 yr] and 8 without deletion [2 females, 6 males; mean age = 18.6 yr, range of 7 to 26 yr]), 21 PLWS parents with an average age of 39.2 yr and a range of 25 to 70 yr (12 fathers [8 fathers of PLWS children with the 15q12 deletion and 4 fathers of PLWS children with normal chromosomes] and 9 mothers [4 mothers of PLWS children with the 15q12 deletion and 5 mothers of PLWS children with normal chromosomes]), and age-matched control individuals. There was no difference between PLWS patients and control individuals in the number of chromosome and chromatid aberrations in cells grown at 48 and/or 96 hr in either 20 ng/ml or 50 ng/ml of MMC or between the PLWS parents and control individuals in cells grown in 50 ng/ml MMC for 96 hr, although a small increase (P less than 0.05) in chromosome breakage was found in cells from the total PLWS parental group compared with control individuals exposed for 48 hr in 50 ng/ml MMC. There was also no significant difference in chromosome fragile site frequency in cells grown in folate-deficient culture conditions in PLWS patients, their parents, or controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fragile sites, Alzheimer's disease, and aging.

The fragile site expression under conditions of folate deprivation was compared in the chromosomes from 5 Alzheimer's disease (AD) female patients, 5 healthy elderly females and 5 healthy young females. Although different fragile sites were observed in the three groups, nevertheless, more similarities were found between the AD patients and elderly normal donors. The only fragile site common to all groups was 3p14. This site was the most frequent in the young donors group. In both AD and elderly control groups we observed a higher frequency of fragility in 6p21, but not in the young controls. Other interesting fragility points observed in these two groups were: 6q21 and 14q24 (in the AD patients) and 9q13, 14q24 and 17q21 (in the healthy aged). 6p21 and 17q21 have been proposed as 'new' fragile sites. We confirm the existence of these fragile sites and comment that in these bands the genes MTBT2 and MTBT1, which are microtubule (beta) associated protein tau-like and tau 1, respectively, are mapped. The tau protein is a component of paired helical filaments which accumulate in degenerating neurons in the brain of patients with AD and with less intensity of normal elderly individuals.     

Enhanced expression of chromosome fragile site 10q25 in chronic myelogenous leukemia

Spontaneous expression of a BrdU-sensitive fragile site at 10q25 was observed in normal lymphocytes and malignant blood and bone marrow cells in chronic myelogenous leukemia (CML). The cells were marked by a Philadelphia chromosome rearrangement due to insertion of 22q11----q13 at 11q13. The fragile site at 10q25 was expressed in larger proportions of malignant than normal cells. Although this fragile site is not at a cancer chromosome breakpoint, malignancy enhanced its expression, consistent with a cascade effect.     

New common fragile sites

We report the finding of a large number of new common fragile sites. Thirty-one (56%) of 55 common fragile sites found in a sample of human lymphocytes were ones not described at the Eighth International Workshop on Human Gene Mapping (HGM 8). The sample consisted of 3023 lymphocytes from nine unrelated individuals with a history of genitourinary malignancy. The lymphocytes were challenged in culture with aphidicolin (Apc), fluorodeoxyuridine (FUdR), 5-azacytidine (Aza), and bromodeoxyuridine (BrdU). Thirteen of the new common fragile sites were induced by Apc and FUdR, nine by Aza, five by BrdU, and four by combined means. The sites induced by Apc and FUdR were cross-induced by BrdU. The fragile sites induced by a diminished concentration of Aza were largely located in heterochromatic regions and were cross-induced by BrdU and FUdR. Exposure to BrdU for 24 hours, a technique hitherto restricted to rare fragile sites, induced several common fragile sites. Control lymphocytes had far fewer gaps and breaks, but these were clustered predominantly at high-expression fragile sites. Because more than half of the common fragile sites in this study were new, it is clear that much remains to be learned. Because the classes of fragile sites reveal cross-induction, we propose that fragile sites share structures in DNA.     

Noninvolvement of chromosome 16 in karyotype evolution of acute myeloid leukemia in a patient with a heritable fragile site on 16q22

A fragile site on the long arm of chromosome #16 (q22) was detected in a 24-year-old man with pancytopenia. During the course of the disease he developed an inverted duplication of region q11-12 of chromosome #1 and a translocation between chromosomes #9 and #13: t(9;13)(p22;q32). These abnormalities, as well as an additional iso-like marker chromosome that consisted of one normal 9p and the abnormal 9p arm, were detected in Epstein-Barr nuclear antigen-positive B-cell cultures. Two years later, evolution of the abnormal clone with loss of chromosome #7 and, subsequently, chromosome #22 occurred in connection with development of acute myeloid leukemia. Although the heritable fragile site on chromosome #16 was present in all cell populations investigated, it was not involved in the evolution of the abnormal karyotype. This fragile chromosome #16 also was found in 4 of 11 family members in whom chromosome analysis was performed, thus suggesting this aberration was inherited in a dominant autosomal pattern. The incidence of the heritable fragile site in normal and leukemic cells of the patient, as well as stimulated blood cultures of his relatives, are reported. In addition, the possible relationship between this constitutional chromosome breakage syndrome and the occurrence of leukemia is analyzed.     

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.     

Constitutive fragile sites 1p31, 3p14, 6q26, and 16q23 and their use as controls for false-negative results with the fragile(X)

Fragile(X) estimations in fragile(X)-mental retardation hemizygotes or heterozygotes can become falsely negative in stored blood (lymphocytes). This was shown in blood stored (before culture) at 4°C, room temperature (25°C), 37°C, and 39°C for 1–4 days. After storage, blood was cultured in Ham's F10-5% FC serum with 0.1 μM FUdR and scored for fra(X) and the constitutive fragile sites at 3p14 and 6q26. It was found that the proportion of cells expressing the fragile(X) and the 3p14 site varied inversely with the temperature and time of storage. In addition, 50 patients and controls were scored for the three latter sites after routine 72–96-hr culture in F 10–0.05 or 0.1 μM FUdR. The 3p14 site was detected in every individual tested in a mean ± S.D. of 11.3 ± 3.2% of cells (0.1 μM FUdR). It was found that this site was FUdR dose dependent whereas the 6q26 site was not. The 3p14 (but not the 6q26) site is therefore suitable as a control site for the FUdR effect. It is proposed that repeat studies are necessary when less then 4% 3p14 sites are present in specimens from males referred for fra(X) estimation. Other constitutive fragile sites (eg, 1p31 and 16q23) can also be used.     

Mutagenicity of algal metabolites of benzo(a)pyrene for Salmonella typhimurium

The metabolism and growth effects of benzo(a)pyrene (BaP) were studied using a freshwater green alga, Selenastrum capricornutum. Algal cultures were incubated under gold light with BaP added at concentrations of 40, 160, 400, and 1,200 micrograms/liter for the periods of 1-4 days. The metabolites and BaP were identified and quantified from ethyl acetate extracts of both algal cells and incubation medium. The ethyl acetate extracts were evaluated for genotoxicity using a micro-volume Salmonella typhimurium forward mutation assay with resistance to 8-azaguanine for selection. This assay detected the presence of small quantities of BaP and was particularly sensitive to the mutagenicity of BaP diols. Of those extracts prepared from algae and medium from cultures exposed to 400 micrograms BaP/liter (10 micrograms/25 ml culture), only algal cell extracts from one day's growth were mutagenic. In cultures exposed to 1,200 micrograms BaP/liter (30 micrograms/25 ml culture), mutagenic materials were produced or persisted in both algae and media throughout the 4-day incubation. The observed mutagenic response can be attributed in part to the presence of unmetabolized BaP or to BaP diols.