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.     

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.     

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.     

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.     

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.     

Chromosome lesions which could be interpreted as "fragile sites" on the distal end of Xq

Chromosome lesions which could be interpreted as "fragile sites" on the distal end of the long arm of the X chromosome were identified during a cytogenetic study of 160 mentally retarded adult males with no apparent cause of their mental retardation and one normal adult female with a family history of fra (X) syndrome. Peripheral blood samples were cultured in either M199 or RPMI 1640 medium with FUdR or BrdU. Metaphases were examined for chromosome lesions or fragile sites on the distal end of Xq and 3 distinct sites were observed: Xq26, Xq27.2, and Xq27.3. Other chromosome lesions at Xq28 were observed and interpreted as nonspecific telomeric structural changes. Chromosome lesions were observed in cells from 14 of the 161 individuals. These included: 5 patients with an Xq26 site, 2 with the recently reported Xq27.2 site, 4 with the Xq27.3 site (characteristic of the fra (X) syndrome), 2 with nonspecific telomeric structural changes, and one individual with 2 lesions (a nonspecific telomeric structural change and an Xq26 site). Additional research is necessary to determine the frequency and clinical significance, if any, of lesions occurring in this region of the X chromosome and to distinguish among heritable fragile sites, constitutive fragile sites, and nonspecific telomeric structural changes.     

Simultaneous expression of the rare and common fragile sites on the X chromosome

The fragile X [fra(X)] syndrome is the most common inherited form of X-linked mental retardation and is associated with a rare folate sensitive fragile site on the X chromosome at band Xq27.3. Recently, a common fragile site located at chromosome band Xq27.2 was delineated (Sutherland & Baker 1990). In order to confirm the previous findings and to further investigate the conditions required for induction of both types of fragile sites, we studied the use of four experimental protocols. Samples from a control male, two fra(X) males and a fra(X) carrier female were studied. Both common and rare fragile sites were seen in the samples from the fra(X) subjects. Up to 4% of cells showed both common and rare fragile sites on the same X chromosome at the 500 band level. The rare and common fragile sites on the X chromosome could be clearly distinguished. From 1 to 3% of the control cells exhibited the common fragile site, while none exhibited the rare fragile site. These protocols should be useful in resolving questionable fra(X) syndrome diagnoses.     

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.     

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.     

Chromosome lesions which could be interpreted as “fragile sites” on the distal end of Xq

Chromosome lesions which could be interpreted as “fragile sites” on the distal end of the long arm of the X chromosome were identified during a cytogenetic study of 160 mentally retarded adult males with no apparent cause of their mental retardation and one normal adult female with a family history of fra (X) syndrome. Peripheral blood samples were cultured in either M199 or RPMI 1640 medium with FUdR or BrdU. Metaphases were examined for chromosome lesions or fragile sites on the distal end of Xq and 3 distinct sites were observed: Xq26, Xq27.2, and Xq27.3. Other chromosome lesions at Xq28 were observed and interpreted as nonspecific telomeric structural changes. Chromosome lesions were observed in cells from 14 of the 161 individuals. These included: 5 patients with an Xq26 site, 2 with the recently reported Xq27.2 site, 4 with the Xq27.3 site (characteristic of the fra (X) syndrome), 2 with nonspecific telomeric structural changes, and one individual with 2 lesions (a nonspecific telomeric structural change and an Xq26 site). Additional research is necessary to determine the frequency and clinical significance, if any, of lesions occurring in this region of the X chromosome and to distinguish among heritable fragile sites, constitutive fragile sites, and nonspecific telomeric structural changes.     

Coincidence in fragile site expression with fluorodeoxyuridine and bromodeoxyuridine

Fragile site expression induced by 10 micrograms/ml or 20 micrograms/ml fluorodeoxyuridine (FudR) and 25 micrograms/ml or 50 micrograms/ml bromodeoxyuridine (BrdU) was studied in lymphocyte cultures of six healthy individuals. A significant decrease in mitotic indexes in respect to control cultures was observed with both FudR concentrations used. The cells showing chromosome aberrations and the total number of cytogenetic alterations were significantly increased both in FudR (p less than 0.001) and BrdU (25 micrograms/ml) (p less than 0.05) treated cultures with respect to the control culture. A site showing a gap or a break was defined as fragile if it appeared in 1% of the cells analyzed and in at least three of the six individuals studied with the same culture treatment. Using these criteria, fragile sites 4q31, 5q15, 6p22, 7p13, 7q32, 13q21, and 14q24 were induced in different proportions by both chemical agents. Although these drugs act via different mechanisms, they both substitute for thymidine in DNA. Our findings suggest that FudR is a more potent common fragile site inducer than BrdU.     

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.     

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).     

In situ nick translation of the fragile X region

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.     

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.     

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.     

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.     

Nature of distamycin A-inducible fragile sites

Five rare distamycin A-inducible fragile sites have been identified on human chromosomes: fra(8)(q24.1), fra(11)(p15.1), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites are located at the junction of Giemsa-positive (G) and negative (R) bands and their expression can be induced by a variety of AT specific DNA ligands. Analysis of family data indicate that the distamycin A-inducible fragile sites segregate as a simple codominant trait with complete penetrance, and probands receive these fragile site genes equally from mothers and fathers. Based on current knowledge of chromosome instability, the nature of distamycin A-inducible fragile sites is discussed. Distamycin A-inducible fragile sites appear to be unique chromosomal regions particularly susceptible to fragility under certain stress conditions. They may also be hot spots for recombination, gene amplification, and integration of foreign genomes.     

Fragile X expression in short-term whole blood cultures is affected by cell density

The effect of cell density on expression of the fragile site at (X)(q27.3) in short-term whole-blood cultures from patients with fragile X [fra(X)] or Martin-Bell syndrome was studied. A significant increase in fra(X) frequency was observed in 7 of 8 samples when cell density was decreased. Higher fra(X) frequency was not always noted at below-routine density, but in some cases fra(X) expression was depressed at above-routine density. We conclude that decay of the FUdR effect explains the fact that fra(X) expression is affected by culture density. It is significant that a relationship exists between the two; it suggests that in order to maximize fra(X) expression in cases with low-percentage fra(X) with standard methods, cell density may have to be adjusted. It is possible that in individuals who are normally nonexpressing, such as some obligate female carriers and nonpenetrant males, fra(X) expression may be sensitive to cell density effects.     

SV40-transformed fragile (X) amniocytes.

We report SV-40 transformation of female and male fragile X [fra(X)] amniocytes. In the transformants, fra(X) (q27.3) was detected in the 7th passage (9 cell generations) in fra(X) female amniocytes. It was conserved until at least the 20th passage (42 cell generations) although the frequency was reduced or became difficult to detect due to karyotypic evolution in the later generations. Similarly, for the male fra(X) amniocyte line, fra(X) was demonstrated at the 2nd passage (3 cell generations) and persisted until at least the 13th passage (29 cell generations). The prolonged period of reproductive potential of these transformed lines ranging from at least 29-42 generations suggests that the cryopreservation of significant quantities of early passage fra(X) transformed amniocytes will assure a reliable and continuous supply of positive control cells. These lines may be used for fra(X) prenatal diagnostic studies thereby improving the ability to quality control the particular fra(X) induction system being used.