Distamycin A-inducible fragile sites and cancer proneness

To determine the baseline frequency of autosomal rare fragile sites in cancer patients, we conducted a population cytogenetic study of 370 patients with leukemias, solid tumors, and other neoplastic disorders. Twenty carriers of rare fragile sites were detected in this patient group. The rare autosomal fragile sites were at fra(8)(q24), fra(11)(p15), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites were found to be distamycin A inducible. Compared with a population incidence in healthy subjects (44 of 845, 5.21%), the overall incidence of distamycin A-inducible fragile sites was not higher in the patient group (20 of 370, 5.41%). Analysis of these individual fragile sites and particular diseases, however, suggests that the distamycin A-inducible fragile sites may play a role in the etiology of leukemia, myeloproliferative disorders, and benign tumors.     

Chromosome abnormalities and rare fragile sites detected in azoospermia patients

Summary We have examined constitutional chromosome abnormalities and fragile sites in 40 patients with azoospermia. Chromosome abnormalities were found in four cases. Three cases showed a deletion of the long arm of the Y chromosome 46,X,del(Yq) and the other case had a ring of G group chromosome 46,XY,r(G). In a rare fragile sites test, four fragile site carriers were detected and three rare autosomal fragile sites were identified; fra(8)(q24.1), fra(11)(p15.1), and fra(17)(p12). The expression of these fragile sites were induced specifically by AT-specific DNA ligands, such as distamycin A and Hoechst 33258. In addition, one patient was found to be the case of double ascertainment of fragile sites, fra(8)(q24.1) and fra(17)(p12). The overall frequency of distamycin A-inducible fragile sites in azoospermia patients appeared to be higher than those reported for Japanese healthy subjects and cancer patients. However, no significant relation among fragile sites, clinical and histological findings has been detected so far.     

A new rare heritable fragile site at 8q24.1 found in a Japanese population

A new rare fragile site, fra(8)(q24.1) (Takahashi et al. 1987), was characterized. This site was confirmed to be heritable from the pedigree analyses of two families. Its expression was induced by AT-specific DNA-ligands: distamycin A, Hoechst 33258, berenil and DAPI, but not in M-F10-, BrdU- and control-cultures. The incidence has already been evaluated to be 0.71% (6/845) in a healthy population (Takahashi et al. 1987). Thus, a fragile 8q24.1 in the present study can be classified into the rare heritable distamycin A-inducible site.     

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.     

Enhancement of a fra(16)(q22) with distamycin A: a family ascertained through an abnormal proposita

A family in which a fragile site at 16q22 was segregating was ascertained through a newborn infant with multiple anomalies. The same fragile site was present in the phenotypically normal father and in a brother with cleft palate. The fra(16)(q22) was similar in appearance, and in response to culture conditions, to that reported by other investigators, including increased breakage in media supplemented with distamycin A. Sampling variation in the frequency of breakage over time may be considerable in some individuals. No pattern of anomalies was found to be associated with the fragile site. However, the reproductive history of the family we report (two livebirths with major congenital anomalies and one stillbirth) suggests caution in concluding fra(16)(q22) is not deleterious.     

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.     

Fragile sites: overview, occurrence in acute nonlymphocytic leukemia and effects of caffeine on expression

Interest has recently grown in the possible role of chromosomal fragile sites as factors predisposing to chromosome rearrangements characteristic of specific human cancers. Data from two series of experiments relating to this hypothesis are presented. First, the effects of caffeine and theophylline on expression of the fragile X and common fragile sites was studied in lymphocytes from three subjects. Caffeine and theophylline did not enhance fragile X expression under the conditions employed but did greatly enhance expression of the common fragile sites. Second, three patients with acute nonlymphocytic leukemia-M4 and inv(16)(p13q22) in leukemic cells were tested for the presence of fra(16)(q22) in normal cells. The fragile site was not seen in any of the patients in this study.     

The inheritance of fragile sites: apparent absence of fra(2)(q13) in the parents of three unrelated probands.

We describe the inherited folate sensitive fragile site, fra(2)(q13), in three unrelated mentally retarded children, two of them with different forms of epilepsy. Fra(2)(q13) was detected in one healthy sib of one of the probands. Except for one cell in one of the fathers, fra(2)(q13) could not be detected in any of the six parents, who were repeatedly studied using methods known to induce fragile sites of this type. These findings suggest that fra(2)(q13) is not associated with the clinical features of our patients and can be transmitted by persons not expressing it. The expression of fra(2)(q13) may be age dependent.     

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.     

Autosomal folate sensitive fragile sites in normal and mentally retarded individuals in Greece

The frequencies of autosomal folate sensitive fragile sites were compared in populations of mentally retarded fra(X) negative (N = 220) and normal children (N = 76) in Greece. In addition, the frequency of autosomal fragile sites was studied in 20 known fra(X) children in order to test if the fra(X) syndrome is associated with general chromosome instability. The frequencies of both common and rare autosomal fragile sites did not differ significantly between the mentally retarded and the normal children, although the rate of expression was considerably higher in the retarded group. Autosomal fragile sites were not increased in the fra(X) patients. Fra(3)(p14) was by far the most frequent one in all groups. The frequency of fra(6)(q26) was found to be considerably higher among the mentally retarded children, this difference being almost statistically significant. Further cytogenetic studies of normal and retarded individuals are required in order to elucidate this point further.     

Spontaneous expression of fra(11)(q23) in a patient with Ewing's sarcoma and t(11;22)(q23;q11)

Cytogenetic analysis of a Ewing's sarcoma revealed a 46,XX,t(8;18)(q11;q21.3), t(11;22)(q23–24;q11–12) chromosome pattern. Observation of t(11;22) is consistent with other reported cases of Ewing's sarcoma. One breakpoint in this translocation, 11q23, coincides with the location of a folate-sensitive fragile site. Examination of peripheral blood leukocyte chromosomes from the patient revealed a 46,XX chromosome pattern with spontaneous, fluorodeoxyuridine-, and Bactrim-induced expression of fra(11)(q23). This may be the first demonstration of constitutional fra(11)(q23) expression in a patient with a neoplasm that exhibits a chromosome rearrangement involving this breakpoint and the first observation of spontaneous expression of this fragile site. These results provide a basis for discussion of the relationship between fragile sites and chromosome rearrangements.     

Fragile (X)(q27) sites in a pedigree with female carriers showing mild to severe mental retardation.

A pedigree showing the fragile site at Xq27 in a severely retarded female and in other less retarded carriers is described. Two of the four moderately retarded males with the fra(X)(q27) show macro-orchidism, and a variety of other features usually used to support the effects of the fra(X)(q27) are also inconsistent. A second fragile site at (10)(q23) is also present and in the two oldest females its frequency is not decreased, whereas the fra(x)(q27) is not detectable in these females although probably present. It is concluded that pedigrees showing mentally retarded females and probable X linkage should be included in studies of the fra(X)(q27).     

Prenatal diagnosis of fragile X syndrome by placental (chorionic villi) biopsy culture

Second trimester ultrasound-guided fetal blood sampling and placental biopsy were performed on 10 pregnancies at risk for fra(X)-linked mental retardation (Martin-Bell syndrome). Three cases were diagnosed as affected after cytogenetic analysis of fetal blood and placental cultures. The fra(X)(q27.3) and common fragile sites were shown to be expressed at a lower level in placenta than in fetal blood. Induction methods included methotrexate, 5-fluoro-2-deoxyuridine, and excess thymidine. Excess thymidine may give the best expression of fra(X)(q27.3). Enhancement of fra(X)(q27.3) expression was not shown with caffeine or 5-methoxybenzamide.     

Normal blood lymphocytes from patients with adipose tissue tumors with rearrangements at 12q13-q14 do not express the fragile site fra(12)(q13.1)

An association between chromosomal fragile sites and cancer-specific breakpoints has been found to be statistically significant. Cancer patients have been shown to be carriers of fragile sites in chromosome regions involved in rearrangements in malignant cells. Based on these observations it has been hypothesized that fragile sites may be involved in the pathogenesis of human tumors. We have recently described a new recurrent cancer breakpoint at chromosomal region 12q13-q14 in adipose tissue tumors. The possible involvement in these tumors of the rare folate-sensitive fragile site 12q13.1 has been investigated in PHA-stimulated peripheral blood cells from three patients carrying the t(12;16)(q13;p11) in their liposarcoma cells and one patient with the t(3;12)(q28;q14) in his lipoma cells. No expression of the fragile site 12q13.1 could be detected in the blood lymphocytes of any of the patients. The involvement of the fragile site 12q13.1 in the pathogenesis of adipose tissue tumors with a 12q13-q14 breakpoint remains to be established.     

Fra(X)(q27.2), the common fragile site, observed in only one of 760 cases studied for the fragile X syndrome.

Cell cultures from 760 whole blood, amniotic fluid, chorionic villus sample, and peripheral umbilical blood sample specimens were exposed to multiple fra(X)(q27.3) induction systems (none had aphidicolin). Fifty-three exhibited the rare fragile site, fra(X)(q27.3) or FRAXA, none of which demonstrated the common fragile site or FRAXD at band Xq27.2. Only one cell in one of the negative whole blood FUdR-treated cultures from a mentally retarded male showed FRAXD. Therefore, it appears that FRAXD occurs very rarely in cultures treated to induce FRAXA since only one positive cell was observed in over 88,000 analyzed. It appears that very low frequencies of fra(X)(q27) can be accounted for only in part by the presence of the common fragile site since only one of 9 cases, each with one fra(X)(q27) positive cell, exhibited FRAXD and the others were FRAXA. After confirmation of FRAXA with direct DNA testing in a large number of low frequency cases, it should be possible to rely on the detection of very low frequencies of fra(X)(q27.3), e.g., 1% with at least 2 positive cells.     

Heritable rare fragile sites in patients with leukemia and other hematologic disorders

Fragile site studies were performed on a total of 126 patients with leukemia and other hematologic disorders including myelodysplastic syndrome (MDS) and polycythemia vera (PV). Compared with an incidence (6.0%) of heritable rare fragile sites in the healthy population, the frequency was not higher in the patient group (3.2%), as a whole. However, two cases of fra(17)(p12) in MDS appeared fourfold larger than expected for this group of patients. In one case, a homozygous carrier of fra(17)(p12) in PV was also very rarely expected from its population incidence. These findings suggested a possible role of rare fragile sites, at least in the etiology of these preleukemic or myeloproliferative disorders.     

Distribution of autosomal fragile sites in specimens cultured for prenatal fragile X diagnosis

We reviewed the distribution of autosomal fragile sites (FS) and spontaneous chromosome breaks or gaps (CB) at chromosome locations other than those recognized as FS from 100 amniotic fluid samples (AF), 19 chorionic villus samples (CVS), and 5 percutaneous umbilical blood samples (PUBS) referred for fragile X [fra(X)] analysis. We present data on the degree of expression of autosomal fragility in AF, CVS, and PUBS samples, and the relationship between degree of expression and induction system. The most common observed FS were: 3p14, 9p32, and 6q26 in AF; 9q32, 3q27, and 8q22 in CVS; and 3p14, Xq22, and 16q23 in PUBS cases. Distribution of FS and CB, when compared by induction system, was not found to be identical. Our data also indicate that the presence of any particular FS cannot be used as an indicator for the effectiveness of the fra(X) induction system in prenatal samples.     

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.     

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.     

Direct intrachromosomal duplication of 16q and heritable fragile site fra (10) (q25) in the same patient

We describe a woman with profound mental retardation and a direct duplication of 16q and fragile site fra(10)(q25). The identification and possible origin of the duplicated 16q is discussed along with the clinical manifestations. To our knowledge this is the first direct duplication of 16q to be reported. The karyotype is shown to be 46,XX, dir dup (16) (q11.2----q13).